RESUMO
In the mouse embryo, the transition from the preimplantation to the postimplantation epiblast is governed by changes in the gene regulatory network (GRN) that lead to transcriptional, epigenetic, and functional changes. This transition can be faithfully recapitulated in vitro by the differentiation of mouse embryonic stem cells (mESCs) to epiblast-like cells (EpiLCs), that reside in naïve and formative states of pluripotency, respectively. However, the GRN that drives this conversion is not fully elucidated. Here we demonstrate that the transcription factor OCT6 is a key driver of this process. Firstly, we show that Oct6 is not expressed in mESCs but is rapidly induced as cells exit the naïve pluripotent state. By deleting Oct6 in mESCs, we find that knockout cells fail to acquire the typical morphological changes associated with the formative state when induced to differentiate. Additionally, the key naïve pluripotency TFs Nanog, Klf2, Nr5a2, Prdm14, and Esrrb were expressed at higher levels than in wild-type cells, indicating an incomplete dismantling of the naïve pluripotency GRN. Conversely, premature expression of Oct6 in naïve cells triggered a rapid morphological transformation mirroring differentiation, that was accompanied by the upregulation of the endogenous Oct6 as well as the formative genes Sox3, Zic2/3, Foxp1, Dnmt3A and FGF5. Strikingly, we found that OCT6 represses Nanog in a bistable manner and that this regulation is at the transcriptional level. Moreover, our findings also reveal that Oct6 is repressed by NANOG. Collectively, our results establish OCT6 as a key TF in the dissolution of the naïve pluripotent state and support a model where Oct6 and Nanog form a double negative feedback loop which could act as an important toggle mediating the transition to the formative state.
Assuntos
Diferenciação Celular , Redes Reguladoras de Genes , Células-Tronco Embrionárias Murinas , Proteína Homeobox Nanog , Animais , Camundongos , Proteína Homeobox Nanog/metabolismo , Proteína Homeobox Nanog/genética , Diferenciação Celular/genética , Células-Tronco Embrionárias Murinas/metabolismo , Células-Tronco Embrionárias Murinas/citologia , Células-Tronco Pluripotentes/metabolismo , Células-Tronco Pluripotentes/citologia , Regulação da Expressão Gênica no Desenvolvimento , Fator 3 de Transcrição de Octâmero/metabolismo , Fator 3 de Transcrição de Octâmero/genética , Camadas Germinativas/metabolismo , Camadas Germinativas/citologia , Camundongos KnockoutRESUMO
Epithelial plasticity involved the terminal and transitional stages that occur during epithelial-to-mesenchymal transition (EMT) and mesenchymal-to-epithelial transition (MET), both are essential at different stages of early embryonic development that have been co-opted by cancer cells to undergo tumor metastasis. These processes are regulated at multiple instances, whereas the post-transcriptional regulation of key genes mediated by microRNAs is gaining major attention as a common and conserved pathway. In this review, we focus on discussing the latest findings of the cellular and molecular basis of the less characterized process of MET during embryonic development, with special attention to the role of microRNAs. Although we take in consideration the necessity of being cautious when extrapolating the obtained evidence, we propose some commonalities between early embryonic development and cancer progression that can shed light into our current understanding of this complex event and might aid in the design of specific therapeutic approaches.
Assuntos
Desenvolvimento Embrionário/genética , Transição Epitelial-Mesenquimal/genética , MicroRNAs/genética , Proteínas de Neoplasias/genética , Neoplasias/genética , Progressão da Doença , Embrião de Mamíferos , Regulação Neoplásica da Expressão Gênica , Camadas Germinativas/citologia , Camadas Germinativas/crescimento & desenvolvimento , Camadas Germinativas/metabolismo , Humanos , MicroRNAs/classificação , MicroRNAs/metabolismo , Metástase Neoplásica , Proteínas de Neoplasias/classificação , Proteínas de Neoplasias/metabolismo , Neoplasias/metabolismo , Neoplasias/patologia , Transdução de Sinais , Somitos/citologia , Somitos/crescimento & desenvolvimento , Somitos/metabolismoRESUMO
BACKGROUND: The segregation of the hypoblast and the emergence of the pluripotent epiblast mark the final stages of blastocyst formation in mammalian embryos. In bovine embryos the formation of the hypoblast has been partially studied, and evidence shows that MEK signalling plays a limited role in the segregation of this lineage. Here we explored the role of different signalling pathways during lineage segregation in the bovine embryo using immunofluorescence analysis of NANOG and SOX17 as readouts of epiblast and hypoblast, respectively. RESULTS: We show that SOX17 starts to be expressed in 16-32-cell stage embryos, whereas NANOG is first detected from 8-cell stage. SOX17 is first co-expressed with NANOG, but these markers become mutually exclusive by the late blastocyst stage. By assessing the expression kinetics of NANOG/SOX17 we show that inhibition of MEK signalling can eliminate SOX17 expression in bovine blastocysts, without altering NANOG expression. Modulation of WNT, PKC and LIF did not affect NANOG expression in the epiblast when used in combination with the ERK inhibitor. CONCLUSIONS: This study shows that SOX17 can be used as a reliable early marker of hypoblast in the bovine, and based on its expression profile we show that the hypoblast segregates in day 7 blastocysts. Furthermore, SOX17 expression is abolished using 1 µM of PD0325901, without affecting the NANOG population in the epiblast. Modulation of WNT, PKC and LIF are not sufficient to support enhanced NANOG expression in the epiblast when combined with ERK inhibitor, indicating that additional signalling pathways should be examined to determine their potential roles in epiblast expansion.
Assuntos
Blastocisto/citologia , Embrião de Mamíferos/embriologia , Camadas Germinativas/embriologia , Proteína Homeobox Nanog/metabolismo , Fatores de Transcrição SOXF/metabolismo , Animais , Benzamidas/farmacologia , Bovinos , Difenilamina/análogos & derivados , Difenilamina/farmacologia , Camadas Germinativas/citologia , Fator Inibidor de Leucemia/biossíntese , Proteína Homeobox Nanog/genética , Proteína Quinase C/biossíntese , Fatores de Transcrição SOXF/genética , Transdução de Sinais/fisiologia , Proteína Wnt1/biossínteseRESUMO
BACKGROUND: Miguel Fernández was an Argentinian zoologist who published the first account of obligate polyembryony in armadillos. His contribution is here discussed in relation to his contemporaries, Newman and Patterson, and more recent work. FINDINGS: Fernandez worked on the mulita (Dasypus hybridus). He was able to get early stages before twinning occurred and show it was preceded by inversion of the germ layers. By the primitive streak stage there were separate embryonic shields and partition of the amnion. There was, however, a single exocoelom and all embryos were enclosed in a common set of membranes comprising chorion towards the attachment site in the uterine fundus and inverted yolk sac on the opposite face. He showed that monozygotic twinning did not occur in another armadillo, the peludo (Chaetophractus villosus). CONCLUSIONS: Fernández's work represented a major breakthrough in understanding how twinning occurred in armadillos. His work and that of others is of intrinsic interest to zoologists and has a direct bearing on the origin of monozygotic twins and birth defects in humans.
Assuntos
Anatomia Comparada/história , Tatus/embriologia , Embriologia/história , Desenvolvimento Embrionário , Camadas Germinativas/embriologia , Gemelaridade Monozigótica , Zoologia/história , Animais , Argentina , Tatus/crescimento & desenvolvimento , Tatus/fisiologia , Membranas Extraembrionárias/citologia , Membranas Extraembrionárias/embriologia , Membranas Extraembrionárias/fisiologia , Feminino , Pesquisa em Genética/história , Camadas Germinativas/citologia , Camadas Germinativas/fisiologia , História do Século XX , Masculino , Placentação , Gravidez , Especificidade da Espécie , Saco Vitelino/citologia , Saco Vitelino/embriologia , Saco Vitelino/fisiologiaRESUMO
BACKGROUND: Comparative studies beyond the traditional model organisms have been instrumental in enhancing our understanding of the conserved and derived features of gastrulation, a fundamental process in which the germ layers are specified and shaped to form the body axis. Here, we analyzed gastrulation in a vertebrate group with an extreme mode of early development, the annual killifish. RESULTS: Gastrulation in annual killifish of the genus Austrolebias takes place after the initially dispersed deep blastomeres congregate to form the so-called reaggregate. Cells from the early reaggregate do not appear to form part of any recognizable axial embryonic structure and are possibly extraembryonic. In contrast, later reaggregate cells become engaged in morphogenetic transformations indicative of a process of gastrulation and axis formation. The expression of brachyury and goosecoid suggests that gastrulation takes place in a compressed blastopore-like structure with an organizer region displaced to one end. No collective cell internalization proper of blastopore architecture is observed, though, and it appears that gastrulation primarily involves the reorganization of individual cells. CONCLUSIONS: The unique mode of gastrulation in annual killifish demonstrates that a process so ancient and fundamental to ontogenesis can have striking morphogenetic variations nonpredicted from the sole examination of model species. Developmental Dynamics 246:812-826, 2017. © 2017 Wiley Periodicals, Inc.
Assuntos
Fundulidae/embriologia , Gastrulação , Camadas Germinativas/citologia , Animais , Embrião não Mamífero , Proteínas Fetais , Fundulidae/fisiologia , Camadas Germinativas/crescimento & desenvolvimento , Camadas Germinativas/metabolismo , Proteína Goosecoid , Proteínas com Domínio TRESUMO
Adult mesenchymal stromal cell-based interventions have shown promising results in a broad range of diseases. However, their use has faced limited effectiveness owing to the low survival rates and susceptibility to environmental stress on transplantation. We describe the cellular and molecular characteristics of multilineage-differentiating stress-enduring (Muse) cells derived from adipose tissue (AT), a subpopulation of pluripotent stem cells isolated from human lipoaspirates. Muse-AT cells were efficiently obtained using a simple, fast, and affordable procedure, avoiding cell sorting and genetic manipulation methods. Muse-AT cells isolated under severe cellular stress, expressed pluripotency stem cell markers and spontaneously differentiated into the three germ lineages. Muse-AT cells grown as spheroids have a limited proliferation rate, a diameter of â¼15 µm, and ultrastructural organization similar to that of embryonic stem cells. Muse-AT cells evidenced high stage-specific embryonic antigen-3 (SSEA-3) expression (â¼60% of cells) after 7-10 days growing in suspension and did not form teratomas when injected into immunodeficient mice. SSEA-3+ -Muse-AT cells expressed CD105, CD29, CD73, human leukocyte antigen (HLA) class I, CD44, and CD90 and low levels of HLA class II, CD45, and CD34. Using lipopolysaccharide-stimulated macrophages and antigen-challenged T-cell assays, we have shown that Muse-AT cells have anti-inflammatory activities downregulating the secretion of proinflammatory cytokines, such as interferon-γ and tumor necrosis factor-α. Muse-AT cells spontaneously gained transforming growth factor-ß1 expression that, in a phosphorylated SMAD2-dependent manner, might prove pivotal in their observed immunoregulatory activity through decreased expression of T-box transcription factor in T cells. Collectively, the present study has demonstrated the feasibility and efficiency of obtaining Muse-AT cells that can potentially be harnessed as immunoregulators to treat immune-related disorders. Stem Cells Translational Medicine 2017;6:161-173.
Assuntos
Tecido Adiposo/patologia , Carcinogênese/patologia , Imunomodulação , Células-Tronco Pluripotentes/citologia , Fator de Crescimento Transformador beta1/farmacologia , Animais , Biomarcadores/metabolismo , Carcinogênese/efeitos dos fármacos , Diferenciação Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Citocinas/metabolismo , Camadas Germinativas/citologia , Humanos , Imunomodulação/efeitos dos fármacos , Cariótipo , Lipopolissacarídeos/farmacologia , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Fosforilação/efeitos dos fármacos , Células-Tronco Pluripotentes/transplante , Células RAW 264.7 , Transdução de Sinais/efeitos dos fármacos , Proteína Smad2/metabolismo , Baço/citologia , Estresse Fisiológico , Teratoma/patologiaRESUMO
In vertebrates, the induction of the three germ layers (ectoderm, mesoderm and endoderm) has been extensively studied, but less is known about how they segregate. Here, we investigated whether Delta-Notch signaling is involved in this process. Activating the pathway in the marginal zone with Notch(ICD) resulted in an expansion of endodermal and neural ectoderm precursors, leaving a thinner mesodermal ring around the blastopore at gastrula stage, when germ layers are segregated. On the other hand, when the pathway was blocked with Delta-1(STU) or with an antisense morpholino oligonucleotide against Notch, the pan-mesodermal brachyury (bra) domain was expanded and the neural border was moved animalwards. Strikingly, the suprablastoporal endoderm was either expanded when Delta-1 signaling was blocked, or reduced after the general knock-down of Notch. In addition, either activating or blocking the pathway delays the blastopore closure. We conclude that the process of delimiting the three germ layers requires Notch signaling, which may be finely regulated by ligands and/or involve non-canonical components of the pathway. Moreover, Notch activity must be modulated at appropriate levels during this process in order to keep normal morphogenetic movements during gastrulation.