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1.
Sci Rep ; 11(1): 14537, 2021 07 15.
Artigo em Inglês | MEDLINE | ID: mdl-34267234

RESUMO

Activin, a member of the transforming growth factor-ß (TGF-ß) superfamily of proteins, induces various tissues from the amphibian presumptive ectoderm, called animal cap explants (ACs) in vitro. However, it remains unclear how and to what extent the resulting cells recapitulate in vivo development. To comprehensively understand whether the molecular dynamics during activin-induced ACs differentiation reflect the normal development, we performed time-course transcriptome profiling of Xenopus ACs treated with 50 ng/mL of activin A, which predominantly induced dorsal mesoderm. The number of differentially expressed genes (DEGs) in response to activin A increased over time, and totally 9857 upregulated and 6663 downregulated DEGs were detected. 1861 common upregulated DEGs among all Post_activin samples included several Spemann's organizer genes. In addition, the temporal transcriptomes were clearly classified into four distinct groups in correspondence with specific features, reflecting stepwise differentiation into mesoderm derivatives, and a decline in the regulation of nuclear envelop and golgi. From the set of early responsive genes, we also identified the suppressor of cytokine signaling 3 (socs3) as a novel activin A-inducible gene. Our transcriptome data provide a framework to elucidate the transcriptional dynamics of activin-driven AC differentiation, reflecting the molecular characteristics of early normal embryogenesis.


Assuntos
Ativinas/farmacologia , Ectoderma/efeitos dos fármacos , Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , Proteínas de Xenopus/genética , Xenopus laevis/embriologia , Animais , Diferenciação Celular/efeitos dos fármacos , Diferenciação Celular/genética , Ectoderma/citologia , Ectoderma/fisiologia , Embrião não Mamífero , Perfilação da Expressão Gênica , Reprodutibilidade dos Testes , Proteína 3 Supressora da Sinalização de Citocinas/genética , Xenopus laevis/genética
2.
PLoS One ; 16(7): e0254024, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34234366

RESUMO

During embryonic development, cells differentiate into a variety of distinct cell types and subtypes with diverse transcriptional profiles. To date, transcriptomic signatures of different cell lineages that arise during development have been only partially characterized. Here we used single-cell RNA-seq to perform transcriptomic analysis of over 20,000 cells disaggregated from the trunk region of zebrafish embryos at the 30 hpf stage. Transcriptional signatures of 27 different cell types and subtypes were identified and annotated during this analysis. This dataset will be a useful resource for many researchers in the fields of developmental and cellular biology and facilitate the understanding of molecular mechanisms that regulate cell lineage choices during development.


Assuntos
Embrião não Mamífero/metabolismo , Perfilação da Expressão Gênica , Análise de Célula Única , Tronco/embriologia , Peixe-Zebra/embriologia , Peixe-Zebra/genética , Animais , Linhagem da Célula/genética , Ectoderma/citologia , Ectoderma/embriologia , Endoderma/citologia , Endoderma/embriologia , Endotélio Vascular/citologia , Endotélio Vascular/embriologia , Eritrócitos/metabolismo , Fibroblastos/citologia , Regulação da Expressão Gênica no Desenvolvimento , Mesoderma/citologia , Mesoderma/embriologia , Músculo Esquelético/embriologia , Músculo Esquelético/metabolismo , Proteínas de Peixe-Zebra/genética , Proteínas de Peixe-Zebra/metabolismo
3.
Nat Commun ; 12(1): 3277, 2021 06 02.
Artigo em Inglês | MEDLINE | ID: mdl-34078907

RESUMO

Generating properly differentiated embryonic structures in vitro from pluripotent stem cells remains a challenge. Here we show that instruction of aggregates of mouse embryonic stem cells with an experimentally engineered morphogen signalling centre, that functions as an organizer, results in the development of embryo-like entities (embryoids). In situ hybridization, immunolabelling, cell tracking and transcriptomic analyses show that these embryoids form the three germ layers through a gastrulation process and that they exhibit a wide range of developmental structures, highly similar to neurula-stage mouse embryos. Embryoids are organized around an axial chordamesoderm, with a dorsal neural plate that displays histological properties similar to the murine embryo neuroepithelium and that folds into a neural tube patterned antero-posteriorly from the posterior midbrain to the tip of the tail. Lateral to the chordamesoderm, embryoids display somitic and intermediate mesoderm, with beating cardiac tissue anteriorly and formation of a vasculature network. Ventrally, embryoids differentiate a primitive gut tube, which is patterned both antero-posteriorly and dorso-ventrally. Altogether, embryoids provide an in vitro model of mammalian embryo that displays extensive development of germ layer derivatives and that promises to be a powerful tool for in vitro studies and disease modelling.


Assuntos
Padronização Corporal/genética , Corpos Embrioides/metabolismo , Desenvolvimento Embrionário/genética , Células-Tronco Embrionárias Murinas/metabolismo , Transdução de Sinais/genética , Animais , Ectoderma/citologia , Ectoderma/crescimento & desenvolvimento , Ectoderma/metabolismo , Embrião de Mamíferos , Corpos Embrioides/citologia , Endoderma/citologia , Endoderma/crescimento & desenvolvimento , Endoderma/metabolismo , Fator de Transcrição GATA6/genética , Fator de Transcrição GATA6/metabolismo , Gástrula/citologia , Gástrula/crescimento & desenvolvimento , Gástrula/metabolismo , Gastrulação/genética , Regulação da Expressão Gênica no Desenvolvimento , Proteínas HMGB/genética , Proteínas HMGB/metabolismo , Camundongos , Células-Tronco Embrionárias Murinas/citologia , Proteína Homeobox Nanog/genética , Proteína Homeobox Nanog/metabolismo , Tubo Neural/citologia , Tubo Neural/crescimento & desenvolvimento , Tubo Neural/metabolismo , Notocorda/citologia , Notocorda/crescimento & desenvolvimento , Notocorda/metabolismo , Fatores de Transcrição SOXF/genética , Fatores de Transcrição SOXF/metabolismo
4.
Development ; 148(10)2021 05 15.
Artigo em Inglês | MEDLINE | ID: mdl-34042967

RESUMO

Regeneration as an adult developmental process is in many aspects similar to embryonic development. Although many studies point out similarities and differences, no large-scale, direct and functional comparative analyses between development and regeneration of a specific cell type or structure in one animal exist. Here, we use the brittle star Amphiura filiformis to characterise the role of the FGF signalling pathway during skeletal development in embryos and arm regeneration. In both processes, we find ligands expressed in ectodermal cells that flank underlying skeletal mesenchymal cells, which express the receptors. Perturbation of FGF signalling showed inhibited skeleton formation in both embryogenesis and regeneration, without affecting other key developmental processes. Differential transcriptome analysis finds mostly differentiation genes rather than transcription factors to be downregulated in both contexts. Moreover, comparative gene analysis allowed us to discover brittle star-specific differentiation genes. In conclusion, our results show that the FGF pathway is crucial for skeletogenesis in the brittle star, as in other deuterostomes, and provide evidence for the re-deployment of a developmental gene regulatory module during regeneration.


Assuntos
Desenvolvimento Ósseo/fisiologia , Regeneração Óssea/fisiologia , Osso e Ossos/embriologia , Fatores de Crescimento de Fibroblastos/metabolismo , Estrelas-do-Mar/embriologia , Animais , Osso e Ossos/metabolismo , Ectoderma/citologia , Ectoderma/metabolismo , Desenvolvimento Embrionário/genética , Extremidades/crescimento & desenvolvimento , Mesoderma/citologia , Mesoderma/metabolismo , Pirróis/farmacologia , Receptor Tipo 1 de Fator de Crescimento de Fibroblastos/antagonistas & inibidores , Transdução de Sinais/fisiologia , Estrelas-do-Mar/genética , Estrelas-do-Mar/metabolismo , Fator A de Crescimento do Endotélio Vascular/metabolismo
5.
Cells ; 10(4)2021 03 30.
Artigo em Inglês | MEDLINE | ID: mdl-33808472

RESUMO

The cells present in the stromal compartment of many tissues are a heterogeneous population containing stem cells, progenitor cells, fibroblasts, and other stromal cells. A SSEA3(+) cell subpopulation isolated from human stromal compartments showed stem cell properties. These cells, known as multilineage-differentiating stress-enduring (MUSE) cells, are capable of resisting stress and possess an excellent ability to repair DNA damage. We isolated MUSE cells from different mouse stromal compartments, such as those present in bone marrow, subcutaneous white adipose tissue, and ear connective tissue. These cells showed overlapping in vitro biological properties. The mouse MUSE cells were positive for stemness markers such as SOX2, OCT3/4, and NANOG. They also expressed TERT, the catalytic telomerase subunit. The mouse MUSE cells showed spontaneous commitment to differentiation in meso/ecto/endodermal derivatives. The demonstration that multilineage stem cells can be isolated from an animal model, such as the mouse, could offer a valid alternative to the use of other stem cells for disease studies and envisage of cellular therapies.


Assuntos
Tecido Adiposo/citologia , Células da Medula Óssea/citologia , Compartimento Celular , Separação Celular , Células do Tecido Conjuntivo/citologia , Orelha/anatomia & histologia , Células-Tronco/citologia , Animais , Biomarcadores/metabolismo , Ciclo Celular , Diferenciação Celular , Ectoderma/citologia , Endoderma/citologia , Mesoderma/citologia , Camundongos Endogâmicos C57BL , Células Estromais/citologia
6.
Nat Commun ; 12(1): 1247, 2021 02 23.
Artigo em Inglês | MEDLINE | ID: mdl-33623021

RESUMO

Extensive epigenetic reprogramming occurs during preimplantation embryo development. However, it remains largely unclear how the drastic epigenetic reprogramming contributes to transcriptional regulatory network during this period. Here, we develop a single-cell multiomics sequencing technology (scNOMeRe-seq) that enables profiling of genome-wide chromatin accessibility, DNA methylation and RNA expression in the same individual cell. We apply this method to depict a single-cell multiomics map of mouse preimplantation development. We find that genome-wide DNA methylation remodeling facilitates the reconstruction of genetic lineages in early embryos. Further, we construct a zygotic genome activation (ZGA)-associated regulatory network and reveal coordination among multiple epigenetic layers, transcription factors and repeat elements that instruct proper ZGA. Cell fates associated cis-regulatory elements are activated stepwise in post-ZGA stages. Trophectoderm (TE)-specific transcription factors play dual roles in promoting the TE program while repressing the inner cell mass (ICM) program during the ICM/TE separation.


Assuntos
Blastocisto/metabolismo , Embrião de Mamíferos/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Genômica , Análise de Célula Única , Alelos , Animais , Linhagem da Célula/genética , Cromatina/metabolismo , Metilação de DNA/genética , Ectoderma/citologia , Feminino , Masculino , Camundongos , Filogenia , Regiões Promotoras Genéticas , Zigoto/metabolismo
7.
Curr Top Dev Biol ; 141: 173-205, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33602488

RESUMO

During the course of evolution, animals have become increasingly complex by the addition of novel cell types and regulatory mechanisms. A prime example is represented by the lateral neural border, known as the neural plate border in vertebrates, a region of the developing ectoderm where presumptive neural and non-neural tissue meet. This region has been intensively studied as the source of two important embryonic cell types unique to vertebrates-the neural crest and the ectodermal placodes-which contribute to diverse differentiated cell types including the peripheral nervous system, pigment cells, bone, and cartilage. How did these multipotent progenitors originate in animal evolution? What triggered the elaboration of the border during the course of chordate evolution? How is the lateral neural border patterned in various bilaterians and what is its fate? Here, we review and compare the development and fate of the lateral neural border in vertebrates and invertebrates and we speculate about its evolutionary origin. Taken together, the data suggest that the lateral neural border existed in bilaterian ancestors prior to the origin of vertebrates and became a developmental source of exquisite evolutionary change that frequently enabled the acquisition of new cell types.


Assuntos
Evolução Biológica , Invertebrados/embriologia , Crista Neural/citologia , Vertebrados/embriologia , Animais , Proteínas Morfogenéticas Ósseas/metabolismo , Cordados não Vertebrados/embriologia , Ectoderma/citologia , Embrião não Mamífero/citologia , Crista Neural/metabolismo , Placa Neural/metabolismo
8.
Dev Biol ; 474: 16-21, 2021 06.
Artigo em Inglês | MEDLINE | ID: mdl-33476596

RESUMO

Recent advances in synthetic human embryology has provided a previously inexistent molecular portrait of human development. Models of synthetic human embryonic tissues capitalize on the self-organizing capabilities of human embryonic stem cells when they are cultured on biomimetic conditions that simulate in vivo human development. In this Review, we discuss these models and how they have shed light on the early stages of human development including amniotic sac development, gastrulation and neurulation. We discuss the mechanisms underlying the molecular logic of embryonic tissue self-organization that have been dissected using synthetic models of human embryology and explore future challenges in the field. Geared with technological advances in bioengineering, high resolution gene expression and imaging tools, these models are set to transform our understanding of the mechanistic basis of embryonic tissue self-organization during human development and how they may go awry in disease.


Assuntos
Desenvolvimento Embrionário , Biologia Sintética/métodos , Âmnio/embriologia , Ectoderma/citologia , Implantação do Embrião , Células-Tronco Embrionárias/citologia , Células-Tronco Embrionárias/fisiologia , Gastrulação , Humanos , Neurulação
9.
Dev Biol ; 470: 74-83, 2021 02.
Artigo em Inglês | MEDLINE | ID: mdl-33159936

RESUMO

We previously identified the protein Lbh as necessary for cranial neural crest (CNC) cell migration in Xenopus through the use of morpholinos. However, Lbh is a maternally deposited protein and morpholinos achieve knockdowns through prevention of translation. In order to investigate the role of Lbh in earlier embryonic events, we employed the new technique "Trim-Away" to degrade this maternally deposited protein. Trim-Away utilizes the E3 ubiquitin ligase trim21 to degrade proteins targeted with an antibody and was developed in mammalian systems. Our results show that Xenopus is amenable to the Trim-Away technique. We also show that early knockdown of Lbh in Xenopus results in defects in gastrulation that present with a decrease in fibronectin matrix assembly, an increased in mesodermal cell migration and decrease in endodermal cell cohesion. We further show that the technique is also effective on a second abundant maternal protein PACSIN2. We discuss potential advantages and limit of the technique in Xenopus embryos as well as the mechanism of gastrulation inhibition.


Assuntos
Gastrulação , Proteínas de Xenopus/fisiologia , Xenopus laevis/embriologia , Proteínas Adaptadoras de Transdução de Sinal/imunologia , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Anticorpos Monoclonais/imunologia , Movimento Celular , Ectoderma/citologia , Ectoderma/embriologia , Ectoderma/patologia , Indução Embrionária , Endoderma/citologia , Endoderma/embriologia , Endoderma/fisiologia , Fibronectinas/metabolismo , Mesoderma/citologia , Mesoderma/embriologia , Mesoderma/fisiologia , Morfolinos , Crista Neural/citologia , Crista Neural/embriologia , Proteólise , Ribonucleoproteínas/genética , Ribonucleoproteínas/metabolismo , Proteínas de Xenopus/genética , Proteínas de Xenopus/imunologia , Proteínas de Xenopus/metabolismo
10.
Cell Tissue Res ; 383(2): 751-763, 2021 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-32960356

RESUMO

Stem cells are a promising tool for treatment of a variety of degenerative diseases. Human amniotic epithelial stem cells (hAECs) have desirable and unique characteristics that make them a proper candidate for cell therapy. In this study, we have investigated the effects of BMP-4 (bone morphogenetic protein-4) and its inhibition on differentiation of AECs into ectodermal lineages. Analysis of AEC-derived ectodermal lineages (neurons and keratinocytes) was performed by using flow cytometry technique for Map2 and ß-tubulin (as neuron markers), Olig2 and MBP (as oligodendrocyte markers), and K14 and K10 (as keratinocyte markers). The results of this study illustrated that noggin (as BMP antagonist), BMP4, and both BMP4 and heparin (together or separately) increased neural and keratinocyte marker expression, respectively. The expression of markers MAP2, olig2, and K14 in hAECs has been significantly decreased 21 days after exposure to differentiation medium (without growth factors) compared with isolation day, which supports the hypothesis that AECs can be dedifferentiated into pluripotent cells. Moreover, activation and inhibition of BMP signaling have no effects on viability of hAECs. The results of this study showed that BMP signaling and its inhibition are the key factors for ectodermal lineage differentiation of amnion-derived stem cells.


Assuntos
Âmnio/citologia , Biomarcadores/metabolismo , Proteínas Morfogenéticas Ósseas/farmacologia , Diferenciação Celular/efeitos dos fármacos , Linhagem da Célula , Ectoderma/citologia , Células Epiteliais/citologia , Células-Tronco/citologia , Desdiferenciação Celular/efeitos dos fármacos , Separação Celular , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/metabolismo , Humanos , Queratinócitos/citologia , Queratinócitos/efeitos dos fármacos , Queratinócitos/metabolismo , Células-Tronco/efeitos dos fármacos , Células-Tronco/metabolismo , Tubulina (Proteína)/metabolismo
11.
Dev Biol ; 470: 84-94, 2021 02.
Artigo em Inglês | MEDLINE | ID: mdl-33217407

RESUMO

At implantation, the mouse embryo undergoes a critical transformation which requires the precise spatiotemporal control of signalling pathways necessary for morphogenesis and developmental progression. The role played by such signalling pathways during this transition are largely unexplored, due to the inaccessibility of the embryo during the implantation when it becomes engulfed by uterine tissues. Genetic studies demonstrate that mutant embryos for BMPs die around gastrulation. Here we have aimed to dissect the role of BMPs during pre-to post-implantation transition by using a protocol permitting the development of the embryo beyond implantation stages in vitro and using stem cells to mimic post-implantation tissue organisation. By assessing both the canonical and non-canonical mechanisms of BMP, we show that the loss of canonical BMP activity compromises the extra-embryonic ectoderm development. Our analyses demonstrate that BMP signalling maintains stem cell populations within both embryonic/extra-embryonic tissues during pre-to post-implantation development. These results may provide insight into the role played by BMP signalling in controlling early embryogenesis.


Assuntos
Proteínas Morfogenéticas Ósseas/metabolismo , Ectoderma/embriologia , Implantação do Embrião , Desenvolvimento Embrionário , Transdução de Sinais , Animais , Morte Celular , Linhagem da Célula , Ectoderma/citologia , Técnicas de Cultura Embrionária , Células-Tronco Embrionárias/citologia , Camadas Germinativas/citologia , Camadas Germinativas/embriologia , Camundongos , Morfogênese , Trofoblastos/citologia
12.
Methods Mol Biol ; 2179: 275-287, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-32939727

RESUMO

Mesenchymal-to-epithelial transition (MET) describes the ability of loosely associated migratory cells to form a more adherent sheet-like assembly of cells. MET is a conserved motif occurring throughout organogenesis and plays a key role in regeneration and cancer metastasis, and is the first step in producing induced pluripotent stem cells (iPSCs). To resolve fundamental biological questions about MET, its relation to epithelial-to-mesenchymal transition, and to explore MET's role in tissue assembly and remodeling requires live models for MET that are amenable to experimentation. Many cases of clinically important MET are inferred since they occur deep with the body of the embryo or adult. We have developed a tractable model for MET, where cellular transitions can be directly observed under conditions where molecular, mechanical, and cellular contexts can be controlled experimentally. In this chapter, we introduce a 3-dimensional (3D) tissue model to study MET using Xenopus laevis embryonic mesenchymal cell aggregates.


Assuntos
Rastreamento de Células/métodos , Ectoderma/citologia , Imageamento Tridimensional/métodos , Mesoderma/citologia , Técnicas de Cultura de Tecidos/métodos , Animais , Movimento Celular , Células Epiteliais/citologia , Células Epiteliais/fisiologia , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/fisiologia , Xenopus
13.
Methods Mol Biol ; 2214: 41-58, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-32944902

RESUMO

Pluripotent stem cells (PSCs) are the in vitro counterpart of the pluripotent epiblast of the mammalian embryo with the capacity to generate all cell types of the adult organism. During development, the three definitive germ layers are specified and simultaneously spatially organized. In contrast, differentiating PSCs tend to generate cell fates in a spatially disorganized manner. This has limited the in vitro study of specific cell-cell interactions and patterning mechanisms that occur in vivo. Here we describe a protocol to differentiate mouse PSCs in a spatially organized manner on micropatterned surfaces. Micropatterned chips comprise many colonies of uniform size and geometry facilitating a robust quantitative analysis of patterned fate specification. Furthermore, multiple factors may be simultaneously manipulated with temporal accuracy to probe the dynamic interactions regulating these processes. The micropattern system is scalable, providing a valuable tool to generate material for large-scale analysis and biochemical experiments that require substantial amounts of starting material, difficult to obtain from early embryos.


Assuntos
Materiais Biocompatíveis/química , Técnicas de Cultura de Células/métodos , Diferenciação Celular , Células-Tronco Pluripotentes/citologia , Animais , Técnicas de Cultura de Células/instrumentação , Células Cultivadas , Ectoderma/citologia , Endoderma/citologia , Desenho de Equipamento , Gastrulação , Camadas Germinativas/citologia , Camundongos , Propriedades de Superfície
14.
Methods Mol Biol ; 2258: 119-130, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33340358

RESUMO

In the developing mammalian embryo, intercellular signaling allows cells to self-organize to create spatial patterns of different cell fates. This process is challenging to study because of the difficulty of observing or manipulating embryos on the spatial and temporal scales required. In vitro models can provide a complement to in vivo systems for addressing these issues. These models are also the only windows we have into early human development. Here we provide protocols for two systems based on differentiating human pluripotent stem cells in micropatterned colonies on defined size and shape. The first model replicates the patterning of the germ layers at gastrulation, while the second replicates the medial-lateral patterning of the ectoderm. These systems allow study of how signaling underlies self-organized patterning at stages of development which are otherwise inaccessible.


Assuntos
Diferenciação Celular , Linhagem da Célula , Ectoderma/fisiologia , Gastrulação , Células-Tronco Embrionárias Humanas/fisiologia , Comunicação Celular , Forma Celular , Tamanho Celular , Células Cultivadas , Ectoderma/citologia , Imunofluorescência , Regulação da Expressão Gênica no Desenvolvimento , Humanos , Microscopia de Fluorescência , Transdução de Sinais , Fatores de Tempo
15.
Int J Dev Biol ; 64(10-11-12): 471-477, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-33336709

RESUMO

FGF signaling pathway is imperative for definitive endoderm (DE) differentiation from human embryonic stem cells (hESCs), which always accompanies an epithelial-to-mesenchymal transition (EMT) process. However, whether there is an association between FGF signaling and the EMT during DE formation in vitro has remained elusive. In the present study, we identify that several FGF family members were significantly activated during the differentiation of hESCs toward DE. Inhibition of FGF signaling by an efficient and selective inhibitor BGJ398 abolishes both the EMT and DE induction by blocking the activation of the zinc-finger transcription factor SNAI1 which is a direct transcriptional repressor of cell adhesion protein CDH1. In addition, cell proliferation is also severely influenced by attenuating the FGF signaling. Collectively, we propose that the FGF signaling promotes the DE formation through mediating the EMT and cell proliferation.


Assuntos
Endoderma/citologia , Transição Epitelial-Mesenquimal , Fatores de Crescimento de Fibroblastos/fisiologia , Transdução de Sinais , Diferenciação Celular , Proliferação de Células , Ectoderma/citologia , Células-Tronco Embrionárias/citologia , Células-Tronco Embrionárias/metabolismo , Transição Epitelial-Mesenquimal/efeitos dos fármacos , Fatores de Crescimento de Fibroblastos/antagonistas & inibidores , Expressão Gênica , Humanos , Compostos de Fenilureia/farmacologia , Pirimidinas/farmacologia
16.
Proc Natl Acad Sci U S A ; 117(52): 33305-33316, 2020 12 29.
Artigo em Inglês | MEDLINE | ID: mdl-33376218

RESUMO

Ectodermal patterning is required for the establishment of multiple components of the vertebrate body plan. Previous studies have demonstrated that precise combinations of extracellular signals induce distinct ectodermal cell populations, such as the neural crest and the neural plate. Yet, we still lack understanding of how the response to inductive signals is modulated to generate the proper transcriptional output in target cells. Here we show that posttranscriptional attenuation of fibroblast growth factor (FGF) signaling is essential for the establishment of the neural crest territory. We found that neural crest progenitors display elevated expression of DICER, which promotes enhanced maturation of a set of cell-type-specific miRNAs. These miRNAs collectively target components of the FGF signaling pathway, a central player in the process of neural induction in amniotes. Inactivation of this posttranscriptional circuit results in a fate switch, in which neural crest cells are converted into progenitors of the central nervous system. Thus, the posttranscriptional attenuation of signaling systems is a prerequisite for proper segregation of ectodermal cell types. These findings demonstrate how posttranscriptional repression may alter the activity of signaling systems to generate distinct spatial domains of progenitor cells.


Assuntos
Fatores de Crescimento de Fibroblastos/genética , Crista Neural/metabolismo , Transdução de Sinais , Transcrição Genética , Animais , Linhagem da Célula , Embrião de Galinha , Ectoderma/citologia , Fatores de Crescimento de Fibroblastos/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , MicroRNAs/genética , MicroRNAs/metabolismo , Modelos Biológicos , Crista Neural/citologia , Ribonuclease III/metabolismo , Transdução de Sinais/genética , Células-Tronco/citologia , Células-Tronco/metabolismo
17.
In Vitro Cell Dev Biol Anim ; 56(10): 859-865, 2020 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-33197035

RESUMO

Spontaneous in vitro hatching of human blastocysts starts with the formation of a tunnel through the zona pellucida (ZP) by cellular projections of trophoblast cells. Our aim was to identify the proteins that are upregulated in these initially hatching cells as compared to trophectoderm (TE) cells from blastocysts that had not yet hatched. Forty seven women that underwent assisted reproduction treatment donated their ICSI-derived polyploid blastocysts for the study. In polyploid blastocysts that started spontaneous hatching, hatched clusters of cells were collected from the outer side of the ZP. Liquid chromatography mass spectrometry was applied to determine the proteins that were upregulated in these cells as compared to TE cells obtained from inside the ZP. Whole non-hatched polyploid blastocysts were used as controls. Overall 1245 proteins were identified in all samples. Forty nine proteins were significantly upregulated in hatching cells and 17 in the TE cells. There was minimal overlap between hatching and TE samples; only serine protease inhibitors (SERPINS) and lipocalin were detected in both samples. Myosin and actin were highly upregulated in the hatching cells as well as paraoxonase, N-acetylmuramoyl alanine amidase, and SERPINS clade A and galectin. In the TE cells, gamma butyrobetaine dioxygenase, lupus La protein, sialidase, lysosomal Pro-X carboxypeptidase, phospholipase b, and SERPINS clade B and A were among the most highly upregulated proteins. These findings may contribute to the basic knowledge of the molecular behavior of the specific cells that actively perforate the glycoprotein matrix of the ZP.


Assuntos
Blastocisto/citologia , Proteômica/métodos , Agregação Celular , Ectoderma/citologia , Humanos , Espectrometria de Massas , Regulação para Cima
18.
Int J Mol Sci ; 21(19)2020 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-33019677

RESUMO

Specification of embryonic lineages is an important question in the field of early development. Numerous studies analyzed the expression patterns of the candidate transcripts and proteins in humans and mice and clearly determined the markers of each lineage. To overcome the limitations of human and mouse embryos, the expression of the marker transcripts in each cell has been investigated using in vivo embryos in pigs. In vitro produced embryos are more accessible, can be rapidly processed with low cost. Therefore, we analyzed the characteristics of lineage markers and the effects of the DAB2 gene (trophectoderm marker) in in vitro fertilized porcine embryos. We investigated the expression levels of the marker genes during embryonic stages and distribution of the marker proteins was assayed in day 7 blastocysts. Then, the shRNA vectors were injected into the fertilized embryos and the differences in the marker transcripts were analyzed. Marker transcripts showed diverse patterns of expression, and each embryonic lineage could be identified with localization of marker proteins. In DAB2-shRNA vectors injected embryos, HNF4A and PDGFRA were upregulated. DAB2 protein level was lower in shRNA-injected embryos without significant differences. Our results will contribute to understanding of the mechanisms of embryonic lineage specification in pigs.


Assuntos
Proteínas Adaptadoras de Transporte Vesicular/genética , Blastocisto/metabolismo , Linhagem da Célula/genética , Ectoderma/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Proteínas Adaptadoras de Transporte Vesicular/antagonistas & inibidores , Proteínas Adaptadoras de Transporte Vesicular/metabolismo , Animais , Biomarcadores/metabolismo , Blastocisto/citologia , Ectoderma/citologia , Ectoderma/crescimento & desenvolvimento , Desenvolvimento Embrionário , Feminino , Fertilização In Vitro , Perfilação da Expressão Gênica , Fator 4 Nuclear de Hepatócito/genética , Fator 4 Nuclear de Hepatócito/metabolismo , Masculino , Oócitos/citologia , Oócitos/metabolismo , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Receptor alfa de Fator de Crescimento Derivado de Plaquetas/genética , Receptor alfa de Fator de Crescimento Derivado de Plaquetas/metabolismo , Espermatozoides/citologia , Espermatozoides/metabolismo , Suínos , Transcrição Genética
19.
Dev Cell ; 55(3): 341-353.e5, 2020 11 09.
Artigo em Inglês | MEDLINE | ID: mdl-33091370

RESUMO

FGF/ERK signaling is crucial for the patterning and proliferation of cell lineages that comprise the mouse blastocyst. However, ERK signaling dynamics have never been directly visualized in live embryos. To address whether differential signaling is associated with particular cell fates and states, we generated a targeted mouse line expressing an ERK-kinase translocation reporter (KTR) that enables live quantification of ERK activity at single-cell resolution. 3D time-lapse imaging of this biosensor in embryos revealed spatially graded ERK activity in the trophectoderm prior to overt polar versus mural differentiation. Within the inner cell mass (ICM), all cells relayed FGF/ERK signals with varying durations and magnitude. Primitive endoderm cells displayed higher overall levels of ERK activity, while pluripotent epiblast cells exhibited lower basal activity with sporadic pulses. These results constitute a direct visualization of signaling events during mammalian pre-implantation development and reveal the existence of spatial and temporal lineage-specific dynamics.


Assuntos
Blastocisto/citologia , Blastocisto/enzimologia , Linhagem da Célula , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Transdução de Sinais , Animais , Sobrevivência Celular , Ectoderma/citologia , Fatores de Crescimento de Fibroblastos/metabolismo , Camundongos , Células-Tronco Embrionárias Murinas/citologia , Fatores de Tempo , Trofoblastos/citologia
20.
Nature ; 587(7834): 443-447, 2020 11.
Artigo em Inglês | MEDLINE | ID: mdl-32968278

RESUMO

Current understandings of cell specification in early mammalian pre-implantation development are based mainly on mouse studies. The first lineage differentiation event occurs at the morula stage, with outer cells initiating a trophectoderm (TE) placental progenitor program. The inner cell mass arises from inner cells during subsequent developmental stages and comprises precursor cells of the embryo proper and yolk sac1. Recent gene-expression analyses suggest that the mechanisms that regulate early lineage specification in the mouse may differ in other mammals, including human2-5 and cow6. Here we show the evolutionary conservation of a molecular cascade that initiates TE segregation in human, cow and mouse embryos. At the morula stage, outer cells acquire an apical-basal cell polarity, with expression of atypical protein kinase C (aPKC) at the contact-free domain, nuclear expression of Hippo signalling pathway effectors and restricted expression of TE-associated factors such as GATA3, which suggests initiation of a TE program. Furthermore, we demonstrate that inhibition of aPKC by small-molecule pharmacological modulation or Trim-Away protein depletion impairs TE initiation at the morula stage. Our comparative embryology analysis provides insights into early lineage specification and suggests that a similar mechanism initiates a TE program in human, cow and mouse embryos.


Assuntos
Evolução Biológica , Ectoderma/metabolismo , Embrião de Mamíferos/citologia , Embrião de Mamíferos/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Transcrição Genética , Trofoblastos/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Massa Celular Interna do Blastocisto/citologia , Massa Celular Interna do Blastocisto/metabolismo , Bovinos , Linhagem da Célula , Polaridade Celular , Ectoderma/citologia , Embrião de Mamíferos/enzimologia , Feminino , Fator de Transcrição GATA3/metabolismo , Humanos , Camundongos , Mórula/citologia , Mórula/enzimologia , Mórula/metabolismo , Placenta/citologia , Placenta/metabolismo , Gravidez , Proteína Quinase C/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Fatores de Transcrição SOXB1/metabolismo , Transdução de Sinais , Fatores de Transcrição/metabolismo , Trofoblastos/citologia , Saco Vitelino/citologia , Saco Vitelino/metabolismo
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