RESUMO
Across the animal kingdom, gastrulation represents a key developmental event during which embryonic pluripotent cells diversify into lineage-specific precursors that will generate the adult organism. Here we report the transcriptional profiles of 116,312 single cells from mouse embryos collected at nine sequential time points ranging from 6.5 to 8.5 days post-fertilization. We construct a molecular map of cellular differentiation from pluripotency towards all major embryonic lineages, and explore the complex events involved in the convergence of visceral and primitive streak-derived endoderm. Furthermore, we use single-cell profiling to show that Tal1-/- chimeric embryos display defects in early mesoderm diversification, and we thus demonstrate how combining temporal and transcriptional information can illuminate gene function. Together, this comprehensive delineation of mammalian cell differentiation trajectories in vivo represents a baseline for understanding the effects of gene mutations during development, as well as a roadmap for the optimization of in vitro differentiation protocols for regenerative medicine.
Assuntos
Diferenciação Celular/genética , Embrião de Mamíferos/citologia , Embrião de Mamíferos/metabolismo , Gastrulação , Organogênese , Análise de Célula Única , Animais , Linhagem da Célula/genética , Quimera/embriologia , Quimera/genética , Quimera/metabolismo , Endoderma/citologia , Endoderma/embriologia , Endoderma/metabolismo , Endotélio/citologia , Endotélio/embriologia , Endotélio/metabolismo , Feminino , Gastrulação/genética , Perfilação da Expressão Gênica , Regulação da Expressão Gênica no Desenvolvimento/genética , Hematopoese/genética , Masculino , Mesoderma/citologia , Mesoderma/embriologia , Camundongos , Mutação/genética , Células Mieloides/citologia , Organogênese/genética , Células-Tronco Pluripotentes/citologia , Células-Tronco Pluripotentes/metabolismo , Linha Primitiva/citologia , Linha Primitiva/embriologia , Proteína 1 de Leucemia Linfocítica Aguda de Células T/deficiência , Proteína 1 de Leucemia Linfocítica Aguda de Células T/genéticaRESUMO
Vascular development begins when mesodermal cells differentiate into endothelial cells, which then form primitive vessels. It has been hypothesized that endothelial-specific gene expression may be regulated combinatorially, but the transcriptional mechanisms governing specificity in vascular gene expression remain incompletely understood. Here, we identify a 44 bp transcriptional enhancer that is sufficient to direct expression specifically and exclusively to the developing vascular endothelium. This enhancer is regulated by a composite cis-acting element, the FOX:ETS motif, which is bound and synergistically activated by Forkhead and Ets transcription factors. We demonstrate that coexpression of the Forkhead protein FoxC2 and the Ets protein Etv2 induces ectopic expression of vascular genes in Xenopus embryos, and that combinatorial knockdown of the orthologous genes in zebrafish embryos disrupts vascular development. Finally, we show that FOX:ETS motifs are present in many known endothelial-specific enhancers and that this motif is an efficient predictor of endothelial enhancers in the human genome.
Assuntos
Elementos Facilitadores Genéticos , Fatores de Transcrição Forkhead/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Proteínas Proto-Oncogênicas c-ets/metabolismo , Animais , Vasos Sanguíneos/embriologia , Embrião de Mamíferos/citologia , Embrião não Mamífero/metabolismo , Endotélio/embriologia , Fibroblastos/metabolismo , Humanos , Camundongos , Xenopus , Peixe-ZebraRESUMO
Branching networks are a very common feature of multicellular animals and underlie the formation and function of numerous organs including the nervous system, the respiratory system, the vasculature and many internal glands. These networks range from subcellular structures such as dendritic trees to large multicellular tissues such as the lungs. The production of branched structures by single cells, so called subcellular branching, which has been better described in neurons and in cells of the respiratory and vascular systems, involves complex cytoskeletal remodelling events. In Drosophila, tracheal system terminal cells (TCs) and nervous system dendritic arborisation (da) neurons are good model systems for these subcellular branching processes. During development, the generation of subcellular branches by single-cells is characterized by extensive remodelling of the microtubule (MT) network and actin cytoskeleton, followed by vesicular transport and membrane dynamics. In this review, we describe the current knowledge on cytoskeletal regulation of subcellular branching, based on the terminal cells of the Drosophila tracheal system, but drawing parallels with dendritic branching and vertebrate vascular subcellular branching.
Assuntos
Diferenciação Celular/fisiologia , Citoesqueleto/fisiologia , Drosophila melanogaster/embriologia , Morfogênese , Neurogênese/fisiologia , Actinas/fisiologia , Animais , Comunicação Celular , Drosophila melanogaster/citologia , Endotélio/embriologia , Humanos , Microtúbulos/fisiologia , Análise de Célula Única , Traqueia/citologia , Traqueia/embriologiaRESUMO
Hematopoietic stem and progenitor cells (HSPCs) in the bone marrow are derived from a small population of hemogenic endothelial (HE) cells located in the major arteries of the mammalian embryo. HE cells undergo an endothelial to hematopoietic cell transition, giving rise to HSPCs that accumulate in intra-arterial clusters (IAC) before colonizing the fetal liver. To examine the cell and molecular transitions between endothelial (E), HE, and IAC cells, and the heterogeneity of HSPCs within IACs, we profiled â¼40 000 cells from the caudal arteries (dorsal aorta, umbilical, vitelline) of 9.5 days post coitus (dpc) to 11.5 dpc mouse embryos by single-cell RNA sequencing and single-cell assay for transposase-accessible chromatin sequencing. We identified a continuous developmental trajectory from E to HE to IAC cells, with identifiable intermediate stages. The intermediate stage most proximal to HE, which we term pre-HE, is characterized by increased accessibility of chromatin enriched for SOX, FOX, GATA, and SMAD motifs. A developmental bottleneck separates pre-HE from HE, with RUNX1 dosage regulating the efficiency of the pre-HE to HE transition. A distal candidate Runx1 enhancer exhibits high chromatin accessibility specifically in pre-HE cells at the bottleneck, but loses accessibility thereafter. Distinct developmental trajectories within IAC cells result in 2 populations of CD45+ HSPCs; an initial wave of lymphomyeloid-biased progenitors, followed by precursors of hematopoietic stem cells (pre-HSCs). This multiomics single-cell atlas significantly expands our understanding of pre-HSC ontogeny.
Assuntos
Diferenciação Celular , Endotélio/embriologia , Hemangioblastos/fisiologia , Hematopoese/fisiologia , Células-Tronco Hematopoéticas/fisiologia , Animais , Diferenciação Celular/genética , Subunidade alfa 2 de Fator de Ligação ao Core/fisiologia , Embrião de Mamíferos , Endotélio/citologia , Endotélio/metabolismo , Feminino , Dosagem de Genes/fisiologia , Regulação da Expressão Gênica no Desenvolvimento , Hemangioblastos/citologia , Hematopoese/genética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Gravidez , RNA-Seq/métodosRESUMO
Hematopoiesis in vertebrate embryos occurs in temporally and spatially overlapping waves in close proximity to blood vascular endothelial cells. Initially, yolk sac hematopoiesis produces primitive erythrocytes, megakaryocytes, and macrophages. Thereafter, sequential waves of definitive hematopoiesis arise from yolk sac and intraembryonic hemogenic endothelia through an endothelial-to-hematopoietic transition (EHT). During EHT, the endothelial and hematopoietic transcriptional programs are tightly co-regulated to orchestrate a shift in cell identity. In the yolk sac, EHT generates erythro-myeloid progenitors, which upon migration to the liver differentiate into fetal blood cells, including erythrocytes and tissue-resident macrophages. In the dorsal aorta, EHT produces hematopoietic stem cells, which engraft the fetal liver and then the bone marrow to sustain adult hematopoiesis. Recent studies have defined the relationship between the developing vascular and hematopoietic systems in animal models, including molecular mechanisms that drive the hemato-endothelial transcription program for EHT. Moreover, human pluripotent stem cells have enabled modeling of fetal human hematopoiesis and have begun to generate cell types of clinical interest for regenerative medicine.
Assuntos
Diferenciação Celular/efeitos da radiação , Linhagem da Célula/fisiologia , Células Endoteliais/metabolismo , Endotélio/embriologia , Hematopoese/fisiologia , Células-Tronco Hematopoéticas/metabolismo , Células Endoteliais/citologia , Endotélio/citologia , Células-Tronco Hematopoéticas/citologia , HumanosRESUMO
Branched structures are evident at all levels of organization in living organisms. Many organs, such as the vascular system, lung, kidney and mammary gland, are heavily branched. In each of these cases, equally fascinating questions have been put forward, including those that address the cellular and molecular mechanisms that regulate the branching process itself, such as where the branches are initiated and how they extend and grow in the right direction. Recent experiments suggest that cell competition and cell rearrangements might be conserved key features in branch formation and might be controlled by local cell signalling.
Assuntos
Endotélio/embriologia , Epitélio/embriologia , Morfogênese , Animais , Humanos , Especificidade de ÓrgãosRESUMO
The endothelium is an additional cell layer, differentiating from the inner epidermis of the ovule integument. In tomato (Solanum lycopersicum L.), after fertilization, the endothelium separates from integument and becomes an independent tissue developing next to the growing embryo sac. In the absence of fertilization, the endothelium may proliferate and form pseudo-embryo. However, the course of the reorganization of endothelium into pseudo-embryo in tomato ovules is poorly understood. We aimed to investigate specific features of endothelium differentiation and the role of the endothelium in the development of fertilized and unfertilized tomato ovules. The ovules of tomato plants ("YaLF" line), produced by vegetative growth plants of transgenic tomato line expressing the ac gene, encoding chitin-binding protein from Amaranthus caudatus L., were investigated using light and transmission electron microscopy. We showed that in the fertilized ovule of normally developing fruit and in the unfertilized ovule of parthenocarpic fruit, separation of the endothelium from integument occurs via programmed death of cells of the integumental parenchyma, adjacent to the endothelium. Endothelial cells in normally developing ovules change their structural and functional specialization from meristematic to secretory and back to meristematic, and proliferate until seeds fully mature. The secretory activity of the endothelium is necessary for the lysis of dying cells of the integument and provides the space for the growth of the new sporophyte. However, in ovules of parthenocarpic fruits, pseudo-embryo cells do not change their structural and functional organization and remain meristematic, no zone of lysis is formed, and pseudo-embryo cells undergo programmed cell death. Our data shows the key role of the endothelium as a protective and secretory tissue, needed for the normal development of ovules.
Assuntos
Endotélio/embriologia , Endotélio/metabolismo , Células Germinativas Vegetais/citologia , Células Germinativas Vegetais/metabolismo , Desenvolvimento Vegetal , Solanum lycopersicum/fisiologia , Diferenciação Celular , Endotélio/citologia , Fertilização , Flores , Regulação da Expressão Gênica de Plantas , Células Germinativas Vegetais/ultraestrutura , Solanum lycopersicum/ultraestrutura , Plantas Geneticamente ModificadasRESUMO
Oxygenated blood from the heart is directed into the systemic circulation through the aortic arch arteries (AAAs). The AAAs arise by remodeling of three symmetrical pairs of pharyngeal arch arteries (PAAs), which connect the heart with the paired dorsal aortae at mid-gestation. Aberrant PAA formation results in defects frequently observed in patients with lethal congenital heart disease. How the PAAs form in mammals is not understood. The work presented in this manuscript shows that the second heart field (SHF) is the major source of progenitors giving rise to the endothelium of the pharyngeal arches 3 - 6, while the endothelium in the pharyngeal arches 1 and 2 is derived from a different source. During the formation of the PAAs 3 - 6, endothelial progenitors in the SHF extend cellular processes toward the pharyngeal endoderm, migrate from the SHF and assemble into a uniform vascular plexus. This plexus then undergoes remodeling, whereby plexus endothelial cells coalesce into a large PAA in each pharyngeal arch. Taken together, our studies establish a platform for investigating cellular and molecular mechanisms regulating PAA formation and alterations that lead to disease.
Assuntos
Região Branquial/embriologia , Endotélio/embriologia , Coração/embriologia , Animais , Aorta/embriologia , Região Branquial/citologia , Sobrevivência Celular , Embrião de Mamíferos/metabolismo , Células Endoteliais/metabolismo , Endotélio/metabolismo , Proteínas de Fluorescência Verde/metabolismo , Camundongos Transgênicos , Células-Tronco/citologia , Fatores de TempoRESUMO
Vessel formation has been extensively studied at the tissue level, but the difficulty in imaging the endothelium with cellular resolution has hampered study of the morphogenesis and behavior of endothelial cells (ECs) in vivo. We are using endothelial-specific transgenes and high-resolution imaging to examine single ECs in zebrafish. By generating mosaics with transgenes that simultaneously mark endothelial nuclei and membranes we are able to definitively identify and study the morphology and behavior of individual ECs during vessel sprouting and lumen formation. Using these methods, we show that developing trunk vessels are composed of ECs of varying morphology, and that single-cell analysis can be used to quantitate alterations in morphology and dynamics in ECs that are defective in proper guidance and patterning. Finally, we use single-cell analysis of intersegmental vessels undergoing lumen formation to demonstrate the coexistence of seamless transcellular lumens and single or multicellular enclosed lumens with autocellular or intercellular junctions, suggesting that heterogeneous mechanisms contribute to vascular lumen formation in vivo. The tools that we have developed for single EC analysis should facilitate further rigorous qualitative and quantitative analysis of EC morphology and behavior in vivo.
Assuntos
Endotélio/citologia , Endotélio/embriologia , Morfogênese , Análise de Célula Única/métodos , Peixe-Zebra/embriologia , Animais , Polaridade Celular , Embrião não Mamífero/citologia , Células Endoteliais/citologia , Endotélio/irrigação sanguínea , Imageamento Tridimensional , Junções Intercelulares , Espaço Intracelular/metabolismo , Fusão de Membrana , Neovascularização Fisiológica , Reprodutibilidade dos Testes , Tronco/irrigação sanguínea , Tronco/embriologia , Vacúolos/metabolismoRESUMO
The transcription factor Runx1 is essential for the formation of yolk sac-derived erythroid/myeloid progenitors (EMPs) and hematopoietic stem cells (HSCs) from hemogenic endothelium during embryogenesis. However, long-term repopulating HSCs (LT-HSCs) persist when Runx1 is conditionally deleted in fetal liver cells, demonstrating that the requirement for Runx1 changes over time. To define more precisely when Runx1 transitions from an essential factor to a homeostatic regulator of EMPs and HSCs, and whether that transition requires fetal liver colonization, we performed conditional, timed deletions of Runx1 between E7.5 and E13.5. We determined that Runx1 loss reduces the formation or function of EMPs up through E10.5. The Runx1 requirement in HSCs ends later, as deletion up to E11.5 eliminates HSCs. At E11.5, there is an abrupt transition to Runx1 independence in at least a subset of HSCs that does not require fetal liver colonization. The transition to Runx1 independence in EMPs is not mediated by other core binding factors (Runx2 and/or Runx3); however, deleting the common non-DNA-binding ß subunit (CBFß) severely compromises LT-HSC function. Hence, the requirements for Runx1 in EMP and HSC formation are temporally distinct, and LT-HSC function is highly reliant on continued core binding factor activity.
Assuntos
Subunidade alfa 2 de Fator de Ligação ao Core/metabolismo , Células-Tronco Hematopoéticas/metabolismo , Animais , Aorta/citologia , Aorta/embriologia , Diferenciação Celular , Galinhas , Ensaio de Unidades Formadoras de Colônias , Embrião de Mamíferos/citologia , Embrião de Mamíferos/metabolismo , Endotélio/embriologia , Endotélio/metabolismo , Epigênese Genética , Células Precursoras Eritroides/citologia , Células Precursoras Eritroides/metabolismo , Feto/embriologia , Deleção de Genes , Células-Tronco Hematopoéticas/citologia , Integrases/metabolismo , Fígado/citologia , Fígado/embriologia , Camundongos , Células Progenitoras Mieloides/citologia , Células Progenitoras Mieloides/metabolismo , Fatores de Tempo , Saco Vitelino/citologiaRESUMO
The developmental relationship between the blood and endothelial cell (EC) lineages remains unclear. In the extra-embryonic blood islands of birds and mammals, ECs and blood cells are closely intermixed, and blood island precursor cells in the primitive streak express many of the same molecular markers, leading to the suggestion that both lineages arise from a common precursor, called the hemangioblast. Cells within the blood island of Xenopus also coexpress predifferentiation markers of the blood and EC lineages. However, using multiple assays, we find that precursor cells in the Xenopus blood island do not normally differentiate into ECs, suggesting that classic hemangioblasts are rare or nonexistent in Xenopus. What prevents these precursor cells from developing into mature ECs? We have found that bone morphogenetic protein (BMP) signaling is essential for erythroid differentiation, and in the absence of BMP signaling, precursor cells adopt an EC fate. Furthermore, inhibition of the erythroid transcription pathway leads to endothelial differentiation. Our results indicate that bipotential endothelial/erythroid precursor cells do indeed exist in the Xenopus blood island, but BMP signaling normally acts to constrain EC fate. More generally, these results provide evidence that commitment to the erythroid lineage limits development of bipotential precursors toward an endothelial fate.
Assuntos
Proteínas Morfogenéticas Ósseas/genética , Células Endoteliais/metabolismo , Endotélio/metabolismo , Células Precursoras Eritroides/metabolismo , Hemangioblastos/metabolismo , Proteínas de Xenopus/genética , Animais , Animais Geneticamente Modificados , Proteínas Morfogenéticas Ósseas/antagonistas & inibidores , Proteínas Morfogenéticas Ósseas/metabolismo , Diferenciação Celular/genética , Linhagem da Célula/genética , Movimento Celular/genética , Embrião não Mamífero/citologia , Embrião não Mamífero/embriologia , Embrião não Mamífero/metabolismo , Células Endoteliais/citologia , Endotélio/citologia , Endotélio/embriologia , Células Precursoras Eritroides/citologia , Eritropoese/genética , Regulação da Expressão Gênica no Desenvolvimento , Técnicas de Silenciamento de Genes , Hemangioblastos/citologia , Sistema Hematopoético/citologia , Sistema Hematopoético/embriologia , Sistema Hematopoético/metabolismo , Hibridização In Situ , Microscopia de Fluorescência , Pirazóis/farmacologia , Pirimidinas/farmacologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética , Proteínas de Xenopus/metabolismo , Xenopus laevis/embriologia , Xenopus laevis/genéticaRESUMO
The vertebrate circulatory system is the most complex vascular system among those of metazoans, with key innovations including a multi-chambered heart and highly specialized blood cells. Invertebrate vessels, on the other hand, consist of hemal spaces between the basal laminae of epithelia. How the evolutionary transition from an invertebrate-type system to the complex vertebrate one occurred is, however, poorly understood. We investigate here the development of the cardiovascular system of the cephalochordate amphioxus Branchiostoma lanceolatum in order to gain insight into the origin of the vertebrate cardiovascular system. The cardiac markers Hand, Csx (Nkx2-5) and Tbx4/5 reveal a broad cardiac-like domain in amphioxus; such a decentralized organization during development parallels that seen in the adult anatomy. Our data therefore support the hypothesis that amphioxus never possessed a proper heart, even transiently during development. We also define a putative hematopoietic domain, supported by the expression of the hematopoietic markers Scl and Pdvegfr. We show that this area is closed to the dorsal aorta anlages, partially linked to excretory tissues, and that its development is regulated by retinoic acid, thus recalling the aorta-gonads-mesonephros (AGM) area of vertebrates. This region probably produces Pdvegfr+ hemal cells, with an important role in amphioxus vessel formation, since treatments with an inhibitor of PDGFR/VEGFR lead to a decrease of Laminin in the basal laminae of developing vessels. Our results point to a chordate origin of hematopoiesis in an AGM-like area from where hemal Pdvegfr+ cells are produced. These Pdvegfr+ cells probably resemble the ancestral chordate blood cells from which the vertebrate endothelium later originated.
Assuntos
Evolução Biológica , Endotélio/embriologia , Hematopoese , Vertebrados/embriologia , Animais , Biomarcadores/metabolismo , Padronização Corporal/efeitos dos fármacos , Padronização Corporal/genética , Sistema Cardiovascular/efeitos dos fármacos , Sistema Cardiovascular/embriologia , Embrião não Mamífero/efeitos dos fármacos , Embrião não Mamífero/metabolismo , Endotélio/efeitos dos fármacos , Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , Hematopoese/efeitos dos fármacos , Hematopoese/genética , Indóis/farmacologia , Larva/efeitos dos fármacos , Larva/genética , Modelos Biológicos , Filogenia , Pirróis/farmacologia , Receptores do Fator de Crescimento Derivado de Plaquetas/antagonistas & inibidores , Receptores do Fator de Crescimento Derivado de Plaquetas/metabolismo , Receptores de Fatores de Crescimento do Endotélio Vascular/antagonistas & inibidores , Receptores de Fatores de Crescimento do Endotélio Vascular/metabolismo , Tretinoína/farmacologia , Vertebrados/genéticaRESUMO
Endothelial-mesenchymal transformation (EMT) is a critical event for the embryonic morphogenesis of cardiac valves. Inducers of EMT during valvulogenesis include VEGF, TGF-ß1, and wnt/ß-catenin (where wnt refers to the wingless-type mammary tumor virus integration site family of proteins), that are regulated in a spatiotemporal manner. EMT has also been observed in diseased, strain-overloaded valve leaflets, suggesting a regulatory role for mechanical strain. Although the preponderance of studies have focused on the role of soluble mitogens, we asked if the valve tissue microenvironment contributed to EMT. To recapitulate these microenvironments in a controlled, in vitro environment, we engineered 2D valve endothelium from sheep valve endothelial cells, using microcontact printing to mimic the regions of isotropy and anisotropy of the leaflet, and applied cyclic mechanical strain in an attempt to induce EMT. We measured EMT in response to both low (10%) and high strain (20%), where low-strain EMT occurred via increased TGF-ß1 signaling and high strain via increased wnt/ß-catenin signaling, suggesting dual strain-dependent routes to distinguish EMT in healthy versus diseased valve tissue. The effect was also directionally dependent, where cyclic strain applied orthogonal to axis of the engineered valve endothelium alignment resulted in severe disruption of cell microarchitecture and greater EMT. Once transformed, these tissues exhibited increased contractility in the presence of endothelin-1 and larger basal mechanical tone in a unique assay developed to measure the contractile tone of the engineered valve tissues. This finding is important, because it implies that the functional properties of the valve are sensitive to EMT. Our results suggest that cyclic mechanical strain regulates EMT in a strain magnitude and directionally dependent manner.
Assuntos
Endotélio/embriologia , Valvas Cardíacas/embriologia , Mesoderma/embriologia , Morfogênese , Estresse Mecânico , Actinas/metabolismo , Animais , Anisotropia , Núcleo Celular/metabolismo , Endotélio/metabolismo , Valvas Cardíacas/metabolismo , Mesoderma/metabolismo , Modelos Biológicos , Contração Miocárdica , Ovinos , Transdução de Sinais , Engenharia TecidualRESUMO
Definitive hematopoietic cells are generated de novo during ontogeny from a specialized subset of endothelium, the so-called hemogenic endothelium. In this review we give a brief overview of the identification of hemogenic endothelium, explore its links with the HSC lineage, and summarize recent insights into the nature of hemogenic endothelium and the microenvironmental and intrinsic regulators contributing to its transition into blood. Ultimately, a better understanding of the processes controlling the transition of endothelium into blood will advance the generation and expansion of hematopoietic stem cells for therapeutic purposes.
Assuntos
Endotélio/fisiologia , Hematopoese/fisiologia , Animais , Linhagem da Célula , Transdiferenciação Celular , Microambiente Celular , Endotélio/embriologia , Células-Tronco Hematopoéticas/citologia , Células-Tronco Hematopoéticas/fisiologia , Humanos , Fatores de Transcrição/metabolismoRESUMO
Cardiac valves originate from endocardial cushions (EC) formed by endothelial-to-mesenchymal transformation (EMT) during embryogenesis. The zinc-finger transcription factor Snai1 has previously been reported to be important for EMT during organogenesis, yet its role in early valve development has not been directly examined. In this study we show that Snai1 is highly expressed in endothelial, and newly transformed mesenchyme cells during EC development. Mice with targeted snai1 knockdown display hypocellular ECs at E10.5 associated with decreased expression of mesenchyme cell markers and downregulation of the matrix metalloproteinase (mmp) family member, mmp15. Snai1 overexpression studies in atrioventricular canal collagen I gel explants indicate that Snai1 is sufficient to promote mmp15 expression, cell transformation, and mesenchymal cell migration and invasion. However, treatment with the catalytically active form of MMP15 promotes cell motility, and not transformation. Further, we show that Snai1-mediated cell migration requires MMP activity, and caMMP15 treatment rescues attenuated migration defects observed in murine ECs following snai1 knockdown. Together, findings from this study reveal previously unappreciated mechanisms of Snai1 for the direct regulation of MMPs during EC development.
Assuntos
Coxins Endocárdicos/metabolismo , Endotélio/metabolismo , Metaloproteinase 15 da Matriz/metabolismo , Mesoderma/metabolismo , Fatores de Transcrição/metabolismo , Animais , Sítios de Ligação/genética , Células COS , Movimento Celular/efeitos dos fármacos , Células Cultivadas , Embrião de Galinha , Chlorocebus aethiops , Dipeptídeos/farmacologia , Coxins Endocárdicos/citologia , Coxins Endocárdicos/embriologia , Endotélio/citologia , Endotélio/embriologia , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Valvas Cardíacas/embriologia , Valvas Cardíacas/metabolismo , Imuno-Histoquímica , Masculino , Metaloproteinase 15 da Matriz/genética , Inibidores de Metaloproteinases de Matriz , Mesoderma/citologia , Mesoderma/embriologia , Camundongos , Camundongos Knockout , Regiões Promotoras Genéticas/genética , Inibidores de Proteases/farmacologia , Ligação Proteica , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fatores de Transcrição da Família Snail , Fatores de Tempo , Fatores de Transcrição/genéticaRESUMO
Notch is a critical mediator of endothelial-to-mesenchymal transition (EndMT) during cardiac cushion development. Slug, a transcriptional repressor that is a Notch target, is an important Notch effector of EndMT in the cardiac cushion. Here, we report that the runt-related transcription factor RUNX3 is a novel direct Notch target in the endothelium. Ectopic expression of RUNX3 in endothelium induces Slug expression and EndMT independent of Notch activation. Interestingly, RUNX3 physically interacts with CSL, the Notch-interacting partner in the nucleus, and induces Slug in a CSL-dependent, but Notch-independent manner. Although RUNX3 may not be required for the initial induction of Slug and EndMT by Notch, because RUNX3 has a much longer half-life than Slug, it sustains the expression of Slug thereby maintaining the mesenchymal phenotype. CSL binds to the Runx3 promoter in the atrioventricular canal in vivo, and inhibition of Notch reduces RUNX3 expression in the cardiac cushion of embryonic hearts. Taken together, our results suggest that induction of RUNX3 may be a mechanism to maintain Notch-transformed mesenchymal cells during heart development.
Assuntos
Subunidade alfa 3 de Fator de Ligação ao Core/metabolismo , Endotélio/embriologia , Transição Epitelial-Mesenquimal/fisiologia , Coração/embriologia , Mesoderma/metabolismo , Regiões Promotoras Genéticas/fisiologia , Receptores Notch/metabolismo , Linhagem Celular , Subunidade alfa 3 de Fator de Ligação ao Core/genética , Embrião de Mamíferos/citologia , Embrião de Mamíferos/embriologia , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Meia-Vida , Humanos , Mesoderma/citologia , Organogênese/fisiologia , Receptores Notch/genética , Fatores de Transcrição da Família Snail , Fatores de Transcrição/biossíntese , Fatores de Transcrição/genéticaRESUMO
BACKGROUND: The signaling cascades that direct the morphological differentiation of the vascular system during early embryogenesis are not well defined. Several signaling pathways, including Notch and VEGF signaling, are critical for the formation of the vasculature in the mouse. To further understand the role of Notch signaling during endothelial differentiation and the genes regulated by this pathway, both loss-of-function and gain-of-function approaches were analyzed in vivo. RESULTS: Conditional transgenic models were used to expand and ablate Notch signaling in the early embryonic endothelium. Embryos with activated Notch1 signaling in the vasculature displayed a variety of defects, and died soon after E10.5. Most notably, the extraembryonic vasculature of the yolk sac displayed remodeling differentiation defects, with greatly enlarged lumens. These phenotypes were distinct from endothelial loss-of-function of RBPJ, a transcriptional regulator of Notch activity. Gene expression analysis of RNA isolated from the yolk sac endothelia of transgenic embryos indicated aberrant expression in a variety of genes in these models. In particular, a variety of secreted factors, including VEGF and TGF-ß family members, displayed coordinate expression defects in the loss-of-function and gain-of-function models. CONCLUSIONS: Morphological analyses of the in vivo models confirm and expand the understanding of Notch signaling in directing endothelial development, specifically in the regulation of vessel diameter in the intra- and extraembryonic vasculature. Expression analysis of these in vivo models suggests that the vascular differentiation defects may be due to the regulation of key genes through the Notch-RBPJ signaling axis. A number of these genes regulated by Notch signaling encode secreted factors, suggesting that Notch signaling may mediate remodeling and vessel diameter in the extraembryonic yolk sac via autocrine and paracrine cell communication. We propose a role for Notch signaling in elaborating the microenvironment of the nascent arteriole, suggesting novel regulatory connections between Notch signaling and other signaling pathways during endothelial differentiation.
Assuntos
Vasos Sanguíneos/embriologia , Receptores Notch/metabolismo , Transdução de Sinais , Saco Vitelino/metabolismo , Animais , Sequência de Bases , Diferenciação Celular , Endotélio/embriologia , Membranas Extraembrionárias/irrigação sanguínea , Membranas Extraembrionárias/metabolismo , Desenvolvimento Fetal , Imunofluorescência , Perfilação da Expressão Gênica , Inativação Gênica , Genótipo , Camundongos/embriologia , Camundongos Transgênicos , Análise em Microsséries , Reação em Cadeia da Polimerase , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Fator de Crescimento Transformador beta/genética , Fator A de Crescimento do Endotélio Vascular/genética , Saco Vitelino/irrigação sanguíneaRESUMO
Blood and endothelium arise in close association during development, possibly from a common precursor, the hemangioblast [1-4]. Genes essential for blood and endothelial development contain functional ETS binding sites, and binding and expression data implicate the transcription factor, friend leukaemia integration 1 (Fli1) [5-10]. However, loss-of-function phenotypes in mice, although suffering both blood and endothelial defects, have thus far precluded the conclusion that Fli1 is essential for these two lineages [11, 12]. By using Xenopus and zebrafish embryos, we show that loss of Fli1 function results in a substantial reduction or absence of hemangioblasts, revealing an absolute requirement. TUNEL assays show that the cells are eventually lost by apoptosis, but only after the regulatory circuit has been disrupted by loss of Fli1. In addition, a constitutively active form of Fli1 is sufficient to induce expression of key hemangioblast genes, such as Scl/Tal1, Lmo2, Gata2, Etsrp, and Flk1. Epistasis assays show that Fli1 expression is induced by Bmp signaling or Cloche, depending on the hemangioblast population, and in both cases Fli1 acts upstream of Gata2, Scl, Lmo2, and Etsrp. Taken together, these results place Fli1 at the top of the transcriptional regulatory hierarchy for hemangioblast specification in vertebrate embryos.
Assuntos
Células Sanguíneas , Endotélio/embriologia , Regulação da Expressão Gênica no Desenvolvimento , Proteína Proto-Oncogênica c-fli-1/metabolismo , Células-Tronco/fisiologia , Animais , Apoptose , Proteínas Morfogenéticas Ósseas/metabolismo , Proteínas de Ligação a DNA/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Fatores de Transcrição/metabolismo , Ativação Transcricional , Proteínas de Xenopus/metabolismo , Xenopus laevis , Peixe-Zebra , Proteínas de Peixe-Zebra/metabolismoRESUMO
SUR-8, a conserved leucine-rich repeats protein, was first identified as a positive regulator of Ras pathway in Caenorhabditis elegans. Biochemical studies indicated that SUR-8 interacts with Ras and Raf, leading to the elevated ERK activity. However, the physiological role of SUR-8 during mammalian development remains unclear. Here we found that germline deletion of SUR-8 in mice resulted in early embryonic lethality. Inactivated SUR-8 specifically in mouse endothelial cells (ECs) revealed that SUR-8 is essential for embryonic heart development. SUR-8 deficiency in ECs resulted in late embryonic lethality, and the mutant mice displayed multiple cardiac defects. The reduced endothelial-mesenchymal transformation (EMT) and the reduced mesenchyme proliferation phase were observed in the atrioventricular canal (AVC) within the mutant hearts, leading to the formation of hypoplastic endocardial cushions. However, ERK activation did not appear to be affected in mutant ECs, suggesting that SUR-8 may act in an ERK-independent pathway to regulate AVC development.