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Origins and Development of the Embryonic Vascular System in XenopusMyers, Candace Tamara January 2013 (has links)
Each step of vascular development needs to be carefully regulated; endothelial precursors must be specified, these cells then proliferate and coalesce to form vascular cords, and finally they lumenate, undergo angiogenic branching and remodeling, and recruit smooth muscle cells to establish a mature vessel. An aberration at any of these steps during embryonic development is incompatible with life, and vascular pathologies in the adult are associated with numerous diseases including stroke, arteriosclerosis, diabetic retinopathies and cancer progression. My work has aimed to understand how endothelial precursors are specified, and more precisely the cell-signaling pathways and transcriptional networks that guide their fate. This work leads us to conclude the following: (1) blood island precursor cells in the Xenopus embryo can give rise to either blood or endothelial cells, and it is BMP-mediated activation of the erythroid transcriptional program that regulates cell fate, (2) endothelial specification requires the Ets transcription factor Etv2. Persistence of Etv2 expression in blood/endothelial cell precursors allows these cells to develop into endothelium, and overexpression of Etv2 in any of the three germ layers causes activation of every endothelial marker examined. Along the way we have characterized a number of small-molecule inhibitors that should be useful to the Xenopus community and applicable to other model systems.
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Characterising Crim1 in Vertebrate DevelopmentGenevieve Kinna Unknown Date (has links)
This thesis investigates the role of Crim1, a transmembrane protein that is expressed in a number of areas in the vertebrate embryo including the developing kidney, eye, testis and spinal cord, which we believe may be a regulator of vertebrate tissue development. To dissect the function of Crim1 in normal mammalian development, two vertebrate models were used, zebrafish and mice. The results show that in zebrafish, crim1 is expressed early in development from the 16-cell stage through to 30 hours post fertilisation (Chapter 3). At 24 hours post fertilisation crim1 is expressed in the intermediate cell mass (icm), the site of haemangioblast development. Haemangioblasts are precursor cells that contribute to the formation of the blood and endothelial cell lineages. Injection of crim1 antisense oligonucleotides into zebrafish embryos (crim1 morphants) lead to an expansion of the icm and defects in the trunk, tail, somites and vasculature. The injection of crim1 antisense oligonucleotides into transgenic fli:GFP zebrafish revealed defects in the intersegmental, dorsal longitudinal anastomotic and parachordal vessels. Although crim1 is expressed during haemagiogensis the primary defect in the crim1 morphant zebrafish appears to be vascular. Further experiments used a ‘knock-in’ mouse, Crim1KST264, in which a loss of functional Crim1 leads to defects in limb (syndactyly), skeleton, eye, vascular, kidney and placental development. Analysis of the kidney phenotype in the embryonic Crim1KST264 homozygotes showed that a loss of Crim1 affects ERK1/2 and phosphorylated-Smad1/5/8 protein expression, although has no direct effect on BMP or TGFβ protein expression (Chapter 4). Analysis of the adult Crim1 outbred kidneys revealed they have albuminuria and leaky vasculature. The complex phenotype presented by the Crim1KST264 homozygote kidneys suggests Crim1 may be regulating multiple growth factor pathways. As Crim1 was shown to be expressed in the placenta, we characterised the role of Crim1 in placental development using the Crim1KST264 mouse (Chapter 5). Crim1KST264 homozygote placentas and embryos are smaller than their wild-type littermates. Our investigations revealed that Crim1 is expressed in trophoblast giant cells and in spongiotrophoblasts. A loss of Crim1 causes a developmental defect in that the junctional zone (region of the placenta containing spongiotrophoblasts and glycogen cells) is expanded, although this phenotype does not appear to be due to a defect in proliferation or apoptosis. Further analysis of E15.5 Crim1KST264 homozygote placentas revealed there was a reduction in the number of labyrinth trophoblast gaint cells. Thus, by using zebrafish and mouse as two model organisms of vertebrate development, this thesis has showed that Crim1 is clearly important for normal embryonic development. To dissect the complex phenotype presented by the Crim1KST264 mouse, further studies of Crim1 and its interaction with other growth factor pathways is needed to elucidate how and to what extent they interact with Crim1 to determine its biological effect on vertebrate tissue.
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Caracterização das Células-Tronco/Progenitoras Hematopoéticas obtidas de Células-Tronco Embrionárias Humanas In Vitro em Sistema de Co-Cultivo com Fibroblastos de Embriões Murinos. / Characterization of Hematopoietic Stem/Progenitor Cells Obtained In Vitro from Human Embryonic Stem Cells in Co-Culture System with Mouse Embryonic Fibroblasts.Costa, Everton de Brito Oliveira 04 June 2012 (has links)
A hematopoese tem sido bem descrita em modelos murinos nas últimas décadas, contudo, trabalhos demonstrando os mecanismos da hematopoese em humanos ainda são escassos. A derivação da primeira linhagem de células-tronco embrionárias humanas (CTEhs) em 1998, gerou novas perspectivas tanto para o estudo da hematopoese na tentativa de mimetizar o que ocorre naturalmente durante o desenvolvimento embrionário, quanto para a aplicação clínica das células hematopoéticas obtidas a partir da diferenciação dessas células. Contudo, apesar de inúmeros trabalhos terem demonstradoa obtenção de células hematopoéticas a partir de CTEhs, os protocolos têm gerado quantidades variáveis de células, com baixa eficiência e com propriedades funcionais de células primitivas. Desse modo, este trabalho procurou estabelecer um modelo próprio de diferenciação de CTEhs-H1 em células progenitoras hematopoéticas para que estas pudessem ser melhor caracterizadas e obtidas de forma mais eficiente. Para isto, foi desenvolvido um sistema de diferenciação baseado no co-cultivo da linhagem de CTEh-H1 com fibroblastos de embrião de camundongo (MEFs), em meio de diferenciação suplementado soro fetal bovino (SFB) e citocinas e fatores de crescimento hematopoéticos em baixas concentrações. Como resultado, o desenvolvimento do presente trabalho permitiu o estabelecimento de um método para geração de populações mistas de células enriquecidas em CPHs positivas para o marcador CD45, o qual mostrou ser coexpresso com outros marcadores hematopoéticos (CD31, CD43, CD71 e CD38), e células hematopoéticas maduras positivas para marcadores mielóide-específicos (235a, CD14, CD15, CD16) e com características morfológicas típicas. Foi demonstrado que as células obtidas expressavam genes relativos ao sistema hematopoético (CD45, CD31, runx1, tal1, lmo2, prom1, CD34 e notch1), e possuíam potencial clonogênico in vitro da ordem de 1/574 células plaqueadas. Em adição, corroboramos os achados de que as células hematopoéticas apresentam duas origens distintas: a partir do endotelio hemogênico e a partir de células com propriedades hemangioblásticas independentes do endotélio hemogênico. / Hematopoiesis has been well described in murine models in recent decades, however, studies demonstrating the mechanisms of hematopoiesis in humans are still scarce. The first human embryonic stem cells line (hESCs) derived in 1998, has generated new perspectives about the study of hematopoiesis as in attempting to mimic what naturally occurs during embryonic development, as for clinical application of hematopoietic cells obtained from the differentiation of these cells. However, although numerous studies have shown the production of hematopoietic cells derived from hESCs, the protocols have generated varying quantities of cells with low efficiency and functional properties of primitive stem cells. Thus, this study sought to establish our own model for hESC-H1 differentiation in hematopoietic progenitor cells so that they could be better characterized and obtained more efficiently. For this way, we developed a differentiation system based on co-culture of hESC-H1 line with inactivated mouse embryonic fibroblasts (MEFs) in differentiation medium supplemented with fetal calf serum (FCS) and cytokines and hematopoietic growth factors in low concentrations. As a result, the development of this study allowed the establishment of a method for generation of mixed population of cells enriched in hematopoietic progenitor cells positive for the marker CD45, which proved to be co-expressed with other hematopoietic markers (CD31, CD43, CD71 and CD38), and mature hematopoietic cells positive for myeloid-specific markers (235a, CD14, CD15, CD16) and morphological characteristics typical. It was shown that these cells expressed genes related to the hematopoietic system (CD45, CD31, runx1, TAL1, LMO2, prom1, CD34 and NOTCH1), and had clonogenic potential in vitro of 1/574 plated cells. In addition, we corroborate the findings that hematopoietic cells have two distinct origins: they can arise as from an hemogenic endothelium as from cells with hemangioblastic properties by an hemogenic endothelium-independent way.
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Caracterização das células-tronco do saco vitelino e análise ultraestrutural da membrana vitelina de embriões ovinos (Ovis aries) / Characterization of stem cells from yolk sac and ultrastructural analysis of the viteline membrane from sheep embryos (Ovis aries)Pessolato, Alícia Greyce Turatti 16 August 2011 (has links)
O saco vitelino é o único anexo embrionário presente em todas as espécies dos embriões vertebrados, répteis, aves e mamíferos. Em mamíferos domésticos o saco vitelino é inicialmente grande, pois nestas espécies ele é transitório. Após a implantação, surge no mesênquima lateral à notocorda agrupamentos de células, denominados ilhotas sanguíneas, que representam os progenitores dos sistemas vascular e hematopoético: os hemangioblastos. Os hemangioblastos centrais das ilhas sanguíneas formam as primeiras células-tronco hematopoéticas, enquanto os hemangioblastos periféricos se diferenciam em angioblastos, os precursores dos vasos sanguíneos. O desenvolvimento inicial da atividade hematopoética no saco vitelino conduz a hipótese de que esse tecido é o local primário de desenvolvimento hematopoético e que as células-tronco derivadas dele semeiam os outros sítios intraembriônicos. Foi possível observar nas análises microscópicas que realmente existe uma relação entre ambas linhagens. Nas análises de expressão gênica, alguns genes expressos pelo hemangioblasto apresentaram alta expressão nas análises D+0 e outros genes também específicos do hemangioblasto, porém em estágios secundários de diferenciação como os encontrados na região aórtica, a nível de endotélio hemogênico apresentaram altos níveis de expressão após 3 dias em cultivo. Concluímos portanto, que o saco vitelino por ser o local primário de formação das células sanguíneas e endoteliais nos estágios iniciais da embriogênese, por serem primitivas e, portanto não expressarem marcadores de células maduras na sua superfície, tornam estas células uma importante fonte de células-tronco relevante para a Terapia Celular para hemofilia e muitas outras doenças humanas. / The yolk sac is the single attachment embryo present in all species of vertebrate embryos, reptiles, birds and mammals. In domestic mammals the yolk sac is initially large, since these species it is transient. After implantation, appears in the lateral mesenchyme to the notochord cell clusters, called \"blood islands\" that represent the progenitors of vascular and hematopoietic systems: the hemangioblasts. The central islands hemangioblasts form the first blood hematopoietic stem cells, while peripheral hemangioblasts, the angioblastic differentiate into the precursors of blood vessels. The initial development of the yolk sac hematopoietic activity leads to the hypothesis that this tissue is the primary site of development and that hematopoietic stem cells derived from them sow other intraembryos sites. It was observed in the microscopic analysis that there is indeed a relationship between the two lineages. In the analysis of gene expression, some genes expressed by hemangioblasts showed high expression in D+0 and other specific genes also hemangioblasts, but in secondary stages of differentiation as found in the aortic region, the level of hemogenic endothelium showed high levels of expression after 3 days in culture. We therefore conclude that the yolk sac to be the primary site of formation of blood and endothelial cells in the early stages of embryogenesis, for its cells be primitive and therefore do not express markers of mature cells on the surface, these cells become an important source of cells relevant to stem cell therapy for hemophilia and many other human diseases.
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Caracterização das Células-Tronco/Progenitoras Hematopoéticas obtidas de Células-Tronco Embrionárias Humanas In Vitro em Sistema de Co-Cultivo com Fibroblastos de Embriões Murinos. / Characterization of Hematopoietic Stem/Progenitor Cells Obtained In Vitro from Human Embryonic Stem Cells in Co-Culture System with Mouse Embryonic Fibroblasts.Everton de Brito Oliveira Costa 04 June 2012 (has links)
A hematopoese tem sido bem descrita em modelos murinos nas últimas décadas, contudo, trabalhos demonstrando os mecanismos da hematopoese em humanos ainda são escassos. A derivação da primeira linhagem de células-tronco embrionárias humanas (CTEhs) em 1998, gerou novas perspectivas tanto para o estudo da hematopoese na tentativa de mimetizar o que ocorre naturalmente durante o desenvolvimento embrionário, quanto para a aplicação clínica das células hematopoéticas obtidas a partir da diferenciação dessas células. Contudo, apesar de inúmeros trabalhos terem demonstradoa obtenção de células hematopoéticas a partir de CTEhs, os protocolos têm gerado quantidades variáveis de células, com baixa eficiência e com propriedades funcionais de células primitivas. Desse modo, este trabalho procurou estabelecer um modelo próprio de diferenciação de CTEhs-H1 em células progenitoras hematopoéticas para que estas pudessem ser melhor caracterizadas e obtidas de forma mais eficiente. Para isto, foi desenvolvido um sistema de diferenciação baseado no co-cultivo da linhagem de CTEh-H1 com fibroblastos de embrião de camundongo (MEFs), em meio de diferenciação suplementado soro fetal bovino (SFB) e citocinas e fatores de crescimento hematopoéticos em baixas concentrações. Como resultado, o desenvolvimento do presente trabalho permitiu o estabelecimento de um método para geração de populações mistas de células enriquecidas em CPHs positivas para o marcador CD45, o qual mostrou ser coexpresso com outros marcadores hematopoéticos (CD31, CD43, CD71 e CD38), e células hematopoéticas maduras positivas para marcadores mielóide-específicos (235a, CD14, CD15, CD16) e com características morfológicas típicas. Foi demonstrado que as células obtidas expressavam genes relativos ao sistema hematopoético (CD45, CD31, runx1, tal1, lmo2, prom1, CD34 e notch1), e possuíam potencial clonogênico in vitro da ordem de 1/574 células plaqueadas. Em adição, corroboramos os achados de que as células hematopoéticas apresentam duas origens distintas: a partir do endotelio hemogênico e a partir de células com propriedades hemangioblásticas independentes do endotélio hemogênico. / Hematopoiesis has been well described in murine models in recent decades, however, studies demonstrating the mechanisms of hematopoiesis in humans are still scarce. The first human embryonic stem cells line (hESCs) derived in 1998, has generated new perspectives about the study of hematopoiesis as in attempting to mimic what naturally occurs during embryonic development, as for clinical application of hematopoietic cells obtained from the differentiation of these cells. However, although numerous studies have shown the production of hematopoietic cells derived from hESCs, the protocols have generated varying quantities of cells with low efficiency and functional properties of primitive stem cells. Thus, this study sought to establish our own model for hESC-H1 differentiation in hematopoietic progenitor cells so that they could be better characterized and obtained more efficiently. For this way, we developed a differentiation system based on co-culture of hESC-H1 line with inactivated mouse embryonic fibroblasts (MEFs) in differentiation medium supplemented with fetal calf serum (FCS) and cytokines and hematopoietic growth factors in low concentrations. As a result, the development of this study allowed the establishment of a method for generation of mixed population of cells enriched in hematopoietic progenitor cells positive for the marker CD45, which proved to be co-expressed with other hematopoietic markers (CD31, CD43, CD71 and CD38), and mature hematopoietic cells positive for myeloid-specific markers (235a, CD14, CD15, CD16) and morphological characteristics typical. It was shown that these cells expressed genes related to the hematopoietic system (CD45, CD31, runx1, TAL1, LMO2, prom1, CD34 and NOTCH1), and had clonogenic potential in vitro of 1/574 plated cells. In addition, we corroborate the findings that hematopoietic cells have two distinct origins: they can arise as from an hemogenic endothelium as from cells with hemangioblastic properties by an hemogenic endothelium-independent way.
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Caracterização das células-tronco do saco vitelino e análise ultraestrutural da membrana vitelina de embriões ovinos (Ovis aries) / Characterization of stem cells from yolk sac and ultrastructural analysis of the viteline membrane from sheep embryos (Ovis aries)Alícia Greyce Turatti Pessolato 16 August 2011 (has links)
O saco vitelino é o único anexo embrionário presente em todas as espécies dos embriões vertebrados, répteis, aves e mamíferos. Em mamíferos domésticos o saco vitelino é inicialmente grande, pois nestas espécies ele é transitório. Após a implantação, surge no mesênquima lateral à notocorda agrupamentos de células, denominados ilhotas sanguíneas, que representam os progenitores dos sistemas vascular e hematopoético: os hemangioblastos. Os hemangioblastos centrais das ilhas sanguíneas formam as primeiras células-tronco hematopoéticas, enquanto os hemangioblastos periféricos se diferenciam em angioblastos, os precursores dos vasos sanguíneos. O desenvolvimento inicial da atividade hematopoética no saco vitelino conduz a hipótese de que esse tecido é o local primário de desenvolvimento hematopoético e que as células-tronco derivadas dele semeiam os outros sítios intraembriônicos. Foi possível observar nas análises microscópicas que realmente existe uma relação entre ambas linhagens. Nas análises de expressão gênica, alguns genes expressos pelo hemangioblasto apresentaram alta expressão nas análises D+0 e outros genes também específicos do hemangioblasto, porém em estágios secundários de diferenciação como os encontrados na região aórtica, a nível de endotélio hemogênico apresentaram altos níveis de expressão após 3 dias em cultivo. Concluímos portanto, que o saco vitelino por ser o local primário de formação das células sanguíneas e endoteliais nos estágios iniciais da embriogênese, por serem primitivas e, portanto não expressarem marcadores de células maduras na sua superfície, tornam estas células uma importante fonte de células-tronco relevante para a Terapia Celular para hemofilia e muitas outras doenças humanas. / The yolk sac is the single attachment embryo present in all species of vertebrate embryos, reptiles, birds and mammals. In domestic mammals the yolk sac is initially large, since these species it is transient. After implantation, appears in the lateral mesenchyme to the notochord cell clusters, called \"blood islands\" that represent the progenitors of vascular and hematopoietic systems: the hemangioblasts. The central islands hemangioblasts form the first blood hematopoietic stem cells, while peripheral hemangioblasts, the angioblastic differentiate into the precursors of blood vessels. The initial development of the yolk sac hematopoietic activity leads to the hypothesis that this tissue is the primary site of development and that hematopoietic stem cells derived from them sow other intraembryos sites. It was observed in the microscopic analysis that there is indeed a relationship between the two lineages. In the analysis of gene expression, some genes expressed by hemangioblasts showed high expression in D+0 and other specific genes also hemangioblasts, but in secondary stages of differentiation as found in the aortic region, the level of hemogenic endothelium showed high levels of expression after 3 days in culture. We therefore conclude that the yolk sac to be the primary site of formation of blood and endothelial cells in the early stages of embryogenesis, for its cells be primitive and therefore do not express markers of mature cells on the surface, these cells become an important source of cells relevant to stem cell therapy for hemophilia and many other human diseases.
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Der Transkriptionsfaktor Hex markiert eine Subpopulation von Endothelzellen bei der Embryonalentwicklung und der Tumorangiogenese / The transscription-factor Hex marks a subpopulation of endothelial cells in embryonic development and in tumor angiogenesisTerwelp, Katrin Elisabeth 16 March 2011 (has links)
No description available.
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