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2.
Nature ; 589(7843): 630-632, 2021 01.
Artigo em Inglês | MEDLINE | ID: mdl-33500572

Assuntos
Anticorpos/uso terapêutico , Biologia Celular , Biologia do Desenvolvimento , Nariz Eletrônico , Espectrometria de Massas/instrumentação , Neurociências , Animais , Anticorpos/química , Anticorpos/genética , Anticorpos/imunologia , Proteínas de Bactérias/efeitos dos fármacos , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/efeitos da radiação , Bioimpressão/tendências , /imunologia , /química , /provisão & distribução , Biologia Celular/instrumentação , Biologia Celular/tendências , Biologia do Desenvolvimento/métodos , Biologia do Desenvolvimento/tendências , Embrião de Mamíferos/citologia , Embrião de Mamíferos/embriologia , Embrião de Mamíferos/metabolismo , Desenvolvimento Embrionário/genética , Holografia/tendências , Humanos , Imunoglobulina E/química , Imunoglobulina E/genética , Imunoglobulina E/imunologia , Imunoglobulina E/uso terapêutico , Canais Iônicos/metabolismo , Espectrometria de Massas/métodos , Proteínas de Membrana/efeitos dos fármacos , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Proteínas de Membrana/efeitos da radiação , Camundongos , Microscopia/instrumentação , Microscopia/tendências , Sondas Moleculares/análise , Neoplasias/tratamento farmacológico , Neurociências/métodos , Neurociências/tendências , Optogenética/tendências , Análise de Célula Única , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz
3.
J Vis Exp ; (162)2020 08 16.
Artigo em Inglês | MEDLINE | ID: mdl-32865527

RESUMO

The goal of this protocol is to establish a robust and reproducible model of α-synuclein accumulation in primary dopamine neurons. Combined with immunostaining and unbiased automated image analysis, this model allows for the analysis of the effects of drugs and genetic manipulations on α-synuclein aggregation in neuronal cultures. Primary midbrain cultures provide a reliable source of bona fide embryonic dopamine neurons. In this protocol, the hallmark histopathology of Parkinson's disease, Lewy bodies (LB), is mimicked by the addition of α-synuclein pre-formed fibrils (PFFs) directly to neuronal culture media. Accumulation of endogenous phosphorylated α-synuclein in the soma of dopamine neurons is detected by immunostaining already at 7 days after the PFF addition. In vitro cell culture conditions are also suitable for the application and evaluation of treatments preventing α-synuclein accumulation, such as small molecule drugs and neurotrophic factors, as well as lentivirus vectors for genetic manipulation (e.g., with CRISPR/Cas9). Culturing the neurons in 96 well plates increases the robustness and power of the experimental setups. At the end of the experiment, the cells are fixed with paraformaldehyde for immunocytochemistry and fluorescence microscopy imaging. Multispectral fluorescence images are obtained via automated microscopy of 96 well plates. These data are quantified (e.g., counting the number of phospho-α-synuclein-containing dopamine neurons per well) with the use of free software that provides a platform for unbiased high-content phenotype analysis. PFF-induced modeling of phosphorylated α-synuclein accumulation in primary dopamine neurons provides a reliable tool to study the underlying mechanisms mediating formation and elimination of α-synuclein inclusions, with the opportunity for high-throughput drug screening and cellular phenotype analysis.


Assuntos
Neurônios Dopaminérgicos/metabolismo , Embrião de Mamíferos/citologia , Mesencéfalo/citologia , alfa-Sinucleína/química , alfa-Sinucleína/metabolismo , Animais , Citoesqueleto/metabolismo , Mesencéfalo/patologia , Camundongos , Doença de Parkinson/metabolismo , Doença de Parkinson/patologia , Agregados Proteicos
4.
Cell Prolif ; 53(11): e12914, 2020 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-32990380

RESUMO

OBJECTIVES: Mouse embryonic stem cell (mESC) culture contains various heterogeneous populations, which serve as excellent models to study gene regulation in early embryo development. The heterogeneity is typically defined by transcriptional activities, for example, the expression of Nanog or Rex1 mRNA. Our objectives were to identify mESC heterogeneity that are caused by mechanisms other than transcriptional control. MATERIALS AND METHODS: Klf3 mRNA and protein were analysed by RT-qPCR, Western blotting or immunofluorescence in mESCs, C2C12 cells, early mouse embryos and various mouse tissues. An ESC reporter line expressing KLF3-GFP fusion protein was made to study heterogeneity of KLF3 protein expression in ESCs. GFP-positive mESCs were sorted for further analysis including RT-qPCR and RNA-seq. RESULTS: In the majority of mESCs, KLF3 protein is actively degraded due to its proline-rich sequence and highly disordered structure. Interestingly, KLF3 protein is stabilized in a small subset of mESCs. Transcriptome analysis indicates that KLF3-positive mESCs upregulate genes that are initially activated in 8-cell embryos. Consistently, KLF3 protein but not mRNA is dramatically increased in 8-cell embryos. Forced expression of KLF3 protein in mESCs promotes the expression of 8-cell-embryo activated genes. CONCLUSIONS: Our study identifies previously unrecognized heterogeneity due to KLF3 protein expression in mESCs.


Assuntos
Regulação da Expressão Gênica no Desenvolvimento , Fatores de Transcrição Kruppel-Like/genética , Células-Tronco Embrionárias Murinas/citologia , Animais , Diferenciação Celular , Linhagem Celular , Embrião de Mamíferos/citologia , Embrião de Mamíferos/metabolismo , Camundongos , Células-Tronco Embrionárias Murinas/metabolismo , Mioblastos/citologia , Mioblastos/metabolismo , RNA Mensageiro/genética , Ativação Transcricional , Transcriptoma
5.
Int J Nanomedicine ; 15: 6201-6209, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32884270

RESUMO

Background: Unique properties of graphene and its derivatives make them attractive in the field of nanomedicine. However, the mass application of graphene might lead to side effects, which has not been properly addressed in previous studies, especially with regard to its effect on the cell cycle. Methods: The effect of two concentrations (100 and 200 µg/mL) of nano- and microsized graphene oxide (nGO and mGO) on apoptosis, cell cycle, and ROS generation was studied. The effect of both sizes on viability and genotoxicity of the embryonic fibroblast cell cycle was evaluated. MTT and flow cytometry were applied to evaluate the effects of graphene oxide (GO) nanosheets on viability of cells. Apoptosis and cell cycle were analyzed by flow cytometry. Results: The results of this study showed that GO disturbed the cell cycle and nGO impaired cell viability by inducing cell apoptosis. Interestingly, both nGO and mGO blocked the cell cycle in the S phase, which is a critical phase of the cell cycle. Upregulation of TP53-gene transcripts was also detected in both nGO- and mGO-treated cells compared to the control, especially at 200 µg/mL. DNA content of the treated cells increased; however, because of DNA degradation, its quality was decreased. Conclusion: In conclusion, graphene oxide at both nano- and micro-scale damages cell physiology and increases cell population in the S phase of the cell cycle.


Assuntos
Ciclo Celular/efeitos dos fármacos , Fibroblastos/efeitos dos fármacos , Grafite/farmacologia , Nanoestruturas/toxicidade , Animais , Apoptose/efeitos dos fármacos , Ciclo Celular/fisiologia , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Embrião de Mamíferos/citologia , Fibroblastos/citologia , Regulação da Expressão Gênica/efeitos dos fármacos , Grafite/toxicidade , Camundongos , Testes de Mutagenicidade , Espécies Reativas de Oxigênio/metabolismo , Proteína Supressora de Tumor p53/genética
6.
Cell Prolif ; 53(10): e12895, 2020 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-32914523

RESUMO

OBJECTIVES: DNA damage and errors of accurate chromosome segregation lead to aneuploidy and foetal defects. DNA repair and the spindle assembly checkpoint (SAC) are the mechanisms developed to protect from these defects. Checkpoint kinase 1 (CHK1) is reported to be an important DNA damage response protein in multiple models, but its functions remain unclear in early mouse embryos. MATERIALS AND METHODS: Immunofluorescence staining, immunoblotting and real-time reverse transcription polymerase chain reaction were used to perform the analyses. Reactive oxygen species levels and Annexin-V were also detected. RESULTS: Loss of CHK1 activity accelerated progress of the cell cycle at the first cleavage; however, it disturbed the development of early embryos to the morula/blastocyst stages. Further analysis indicated that CHK1 participated in spindle assembly and chromosome alignment, possibly due to its regulation of kinetochore-microtubule attachment and recruitment of BubR1 and p-Aurora B to the kinetochores, indicating its role in SAC activity. Loss of CHK1 activity led to embryonic DNA damage and oxidative stress, which further induced early apoptosis and autophagy, indicating that CHK1 is responsible for interphase DNA damage repair. CONCLUSIONS: Our results indicate that CHK1 is a key regulator of the SAC and DNA damage repair during early embryonic development in mice.


Assuntos
Quinase 1 do Ponto de Checagem/metabolismo , Reparo do DNA , Pontos de Checagem da Fase M do Ciclo Celular , Animais , Apoptose/efeitos dos fármacos , Aurora Quinase B/metabolismo , Proteínas de Ciclo Celular/metabolismo , Quinase 1 do Ponto de Checagem/antagonistas & inibidores , Segregação de Cromossomos/efeitos dos fármacos , Dano ao DNA/efeitos dos fármacos , Embrião de Mamíferos/citologia , Embrião de Mamíferos/metabolismo , Desenvolvimento Embrionário/efeitos dos fármacos , Cinetocoros/metabolismo , Pontos de Checagem da Fase M do Ciclo Celular/efeitos dos fármacos , Camundongos , Microtúbulos/metabolismo , Estresse Oxidativo/efeitos dos fármacos , Compostos de Fenilureia/farmacologia , Proteínas Serina-Treonina Quinases/metabolismo , Pirazinas/farmacologia , Espécies Reativas de Oxigênio/metabolismo
7.
J Vis Exp ; (163)2020 09 09.
Artigo em Inglês | MEDLINE | ID: mdl-32986023

RESUMO

Parkinson's disease (PD) is a devastating neurodegenerative disorder caused by the degeneration of dopaminergic (DA) neurons. Excessive Ca2+ influx due to the abnormal activation of glutamate receptors results in DA excitotoxicity and has been identified as an important mechanism for DA neuron loss. In this study, we isolate, dissociate, and culture midbrain neurons from the mouse ventral mesencephalon (VM) of ED14 mouse embryos. We then infect the long-term primary mouse midbrain cultures with an adeno-associated virus (AAV) expressing a genetically encoded calcium indicator, GCaMP6f under control of the human neuron-specific synapsin promoter, hSyn. Using live confocal imaging, we show that cultured mouse midbrain neurons display spontaneous Ca2+ fluxes detected by AAV-hSyn-GCaMP6f. Bath application of glutamate to midbrain cultures causes abnormal elevations in intracellular Ca2+ within neurons and this is accompanied by caspase-3 activation in DA neurons, as demonstrated by immunostaining. The techniques to identify glutamate-mediated apoptosis in primary mouse DA neurons have important applications for the high content screening of drugs that preserve DA neuron health.


Assuntos
Sinalização do Cálcio , Cálcio/metabolismo , Neurônios Dopaminérgicos/citologia , Mesencéfalo/citologia , Animais , Caspase 3/metabolismo , Células Cultivadas , Dependovirus/genética , Neurônios Dopaminérgicos/efeitos dos fármacos , Embrião de Mamíferos/citologia , Vetores Genéticos/metabolismo , Glutamatos/farmacologia , Processamento de Imagem Assistida por Computador , Camundongos , Quinoxalinas/farmacologia , Receptores de AMPA/metabolismo , Tirosina 3-Mono-Oxigenase/metabolismo
8.
Nature ; 585(7825): 404-409, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32848249

RESUMO

To implant in the uterus, the mammalian embryo first specifies two cell lineages: the pluripotent inner cell mass that forms the fetus, and the outer trophectoderm layer that forms the placenta1. In many organisms, asymmetrically inherited fate determinants drive lineage specification2, but this is not thought to be the case during early mammalian development. Here we show that intermediate filaments assembled by keratins function as asymmetrically inherited fate determinants in the mammalian embryo. Unlike F-actin or microtubules, keratins are the first major components of the cytoskeleton that display prominent cell-to-cell variability, triggered by heterogeneities in the BAF chromatin-remodelling complex. Live-embryo imaging shows that keratins become asymmetrically inherited by outer daughter cells during cell division, where they stabilize the cortex to promote apical polarization and YAP-dependent expression of CDX2, thereby specifying the first trophectoderm cells of the embryo. Together, our data reveal a mechanism by which cell-to-cell heterogeneities that appear before the segregation of the trophectoderm and the inner cell mass influence lineage fate, via differential keratin regulation, and identify an early function for intermediate filaments in development.


Assuntos
Linhagem da Célula , Embrião de Mamíferos/citologia , Embrião de Mamíferos/metabolismo , Queratinas/metabolismo , Actinas/metabolismo , Animais , Divisão Celular , Montagem e Desmontagem da Cromatina , Proteínas Cromossômicas não Histona/metabolismo , Ectoderma/citologia , Embrião de Mamíferos/embriologia , Feminino , Humanos , Filamentos Intermediários/metabolismo , Camundongos , Microtúbulos/metabolismo , Complexos Multiproteicos/metabolismo , Trofoblastos/citologia
9.
Nature ; 583(7818): 760-767, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32728245

RESUMO

During mammalian embryogenesis, differential gene expression gradually builds the identity and complexity of each tissue and organ system1. Here we systematically quantified mouse polyA-RNA from day 10.5 of embryonic development to birth, sampling 17 tissues and organs. The resulting developmental transcriptome is globally structured by dynamic cytodifferentiation, body-axis and cell-proliferation gene sets that were further characterized by the transcription factor motif codes of their promoters. We decomposed the tissue-level transcriptome using single-cell RNA-seq (sequencing of RNA reverse transcribed into cDNA) and found that neurogenesis and haematopoiesis dominate at both the gene and cellular levels, jointly accounting for one-third of differential gene expression and more than 40% of identified cell types. By integrating promoter sequence motifs with companion ENCODE epigenomic profiles, we identified a prominent promoter de-repression mechanism in neuronal expression clusters that was attributable to known and novel repressors. Focusing on the developing limb, single-cell RNA data identified 25 candidate cell types that included progenitor and differentiating states with computationally inferred lineage relationships. We extracted cell-type transcription factor networks and complementary sets of candidate enhancer elements by using single-cell RNA-seq to decompose integrative cis-element (IDEAS) models that were derived from whole-tissue epigenome chromatin data. These ENCODE reference data, computed network components and IDEAS chromatin segmentations are companion resources to the matching epigenomic developmental matrix, and are available for researchers to further mine and integrate.


Assuntos
Embrião de Mamíferos/citologia , Embrião de Mamíferos/embriologia , Desenvolvimento Embrionário/genética , Regulação da Expressão Gênica no Desenvolvimento , Análise de Célula Única , Transcriptoma , Animais , Diferenciação Celular/genética , Linhagem da Célula/genética , Cromatina/genética , Embrião de Mamíferos/metabolismo , Elementos Facilitadores Genéticos , Epigenômica , Extremidades/embriologia , Feminino , Masculino , Camundongos , Poli A/genética , Poli A/metabolismo , Regiões Promotoras Genéticas , RNA-Seq , Fatores de Transcrição/metabolismo
10.
Nat Cell Biol ; 22(7): 767-778, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32601371

RESUMO

Following fertilization in mammals, the gametes are reprogrammed to create a totipotent zygote, a process that involves de novo establishment of chromatin domains. A major feature occurring during preimplantation development is the dramatic remodelling of constitutive heterochromatin, although the functional relevance of this is unknown. Here, we show that heterochromatin establishment relies on the stepwise expression and regulated activity of SUV39H enzymes. Enforcing precocious acquisition of constitutive heterochromatin results in compromised development and epigenetic reprogramming, which demonstrates that heterochromatin remodelling is essential for natural reprogramming at fertilization. We find that de novo H3K9 trimethylation (H3K9me3) in the paternal pronucleus after fertilization is catalysed by SUV39H2 and that pericentromeric RNAs inhibit SUV39H2 activity and reduce H3K9me3. De novo H3K9me3 is initially non-repressive for gene expression, but instead bookmarks promoters for compaction. Overall, we uncover the functional importance for the restricted transmission of constitutive heterochromatin during reprogramming and a non-repressive role for H3K9me3.


Assuntos
Centrômero/genética , Embrião de Mamíferos/citologia , Embrião de Mamíferos/metabolismo , Desenvolvimento Embrionário , Heterocromatina/metabolismo , Histonas/metabolismo , RNA/metabolismo , Animais , Núcleo Celular/genética , Núcleo Celular/metabolismo , Epigênese Genética , Feminino , Heterocromatina/genética , Histonas/genética , Masculino , Metilação , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos CBA , RNA/genética
11.
PLoS One ; 15(7): e0223633, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32701951

RESUMO

BACKGROUND: Small conductance, calcium-activated (SK3) potassium channels control the intrinsic excitability of dopaminergic neurons (DN) in the midbrain and modulate their susceptibility to toxic insults during development. METHODS: We evaluated the age-dependency of the neuroprotective effect of an SK3 agonist, 1-Ethyl-1,3-dihydro-2H-benzimidazol-2-one (1-EBIO), on Amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) excitotoxicity to DN in ventral mesencephalon (VM) organotypic cultures. RESULTS: Most tyrosine hydroxylase (TH)+ neurons were also SK3+; SK3+/TH- cells (DN+) were common at each developmental stage but more prominently at day in vitro (DIV) 8. Young DN+ neurons were small bipolar and fusiform, whereas mature ones were large and multipolar. Exposure of organotypic cultures to AMPA (100 µm, 16 h) had no effect on the survival of DN+ at DIV 8, but caused significant toxicity at DIV 15 (n = 15, p = 0.005) and DIV 22 (n = 15, p<0.001). These results indicate that susceptibility of DN to AMPA excitotoxicity is developmental stage-dependent in embryonic VM organotypic cultures. Immature DN+ (small, bipolar) were increased after AMPA (100 µm, 16 h) at DIV 8, at the expense of the number of differentiated (large, multipolar) DN+ (p = 0.039). This effect was larger at DIV 15 (p<<<0.0001) and at DIV 22 (p<<<0.0001). At DIV 8, 30 µM 1-EBIO resulted in a large increase in DN+. At DIV 15, AMPA toxicity was prevented by exposure to 30 µM, but not 100 µM 1-EBIO. At DIV 22, excitotoxicity was unaffected by 30 µM 1-EBIO, and partially reduced by 100 µM 1-EBIO. CONCLUSION: The effects of the SK3 channel agonist 1-EBIO on the survival of SK3-expressing dopaminergic neurons were concentration-dependent and influenced by neuronal developmental stage.


Assuntos
Neurônios Dopaminérgicos/efeitos dos fármacos , Fármacos Neuroprotetores/farmacologia , Canais de Potássio Ativados por Cálcio de Condutância Baixa/agonistas , Animais , Benzimidazóis/farmacologia , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Neurônios Dopaminérgicos/metabolismo , Neurônios Dopaminérgicos/patologia , Embrião de Mamíferos/citologia , Desenvolvimento Embrionário/efeitos dos fármacos , Feminino , Mesencéfalo/citologia , Mesencéfalo/metabolismo , Ratos , Ratos Sprague-Dawley , Receptores de AMPA/metabolismo , Ácido alfa-Amino-3-hidroxi-5-metil-4-isoxazol Propiônico/toxicidade
12.
Nature ; 582(7811): 253-258, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-32523119

RESUMO

Tissue sculpting during development has been attributed mainly to cellular events through processes such as convergent extension or apical constriction1,2. However, recent work has revealed roles for basement membrane remodelling in global tissue morphogenesis3-5. Upon implantation, the epiblast and extraembryonic ectoderm of the mouse embryo become enveloped by a basement membrane. Signalling between the basement membrane and these tissues is critical for cell polarization and the ensuing morphogenesis6,7. However, the mechanical role of the basement membrane in post-implantation embryogenesis remains unknown. Here we demonstrate the importance of spatiotemporally regulated basement membrane remodelling during early embryonic development. Specifically, we show that Nodal signalling directs the generation and dynamic distribution of perforations in the basement membrane by regulating the expression of matrix metalloproteinases. This basement membrane remodelling facilitates embryo growth before gastrulation. The establishment of the anterior-posterior axis8,9 further regulates basement membrane remodelling by localizing Nodal signalling-and therefore the activity of matrix metalloproteinases and basement membrane perforations-to the posterior side of the embryo. Perforations on the posterior side are essential for primitive-streak extension during gastrulation by rendering the basement membrane of the prospective primitive streak more prone to breaching. Thus spatiotemporally regulated basement membrane remodelling contributes to the coordination of embryo growth, morphogenesis and gastrulation.


Assuntos
Membrana Basal/embriologia , Membrana Basal/metabolismo , Embrião de Mamíferos/embriologia , Embrião de Mamíferos/metabolismo , Desenvolvimento Embrionário , Animais , Membrana Basal/citologia , Blastocisto/citologia , Blastocisto/metabolismo , Embrião de Mamíferos/citologia , Matriz Extracelular/metabolismo , Feminino , Gástrula/embriologia , Masculino , Metaloproteinases da Matriz/metabolismo , Camundongos , Ligantes da Sinalização Nodal/metabolismo , Linha Primitiva/citologia , Linha Primitiva/embriologia , Linha Primitiva/metabolismo
13.
Nature ; 582(7813): 571-576, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-32499656

RESUMO

Macrophages are the first cells of the nascent immune system to emerge during embryonic development. In mice, embryonic macrophages infiltrate developing organs, where they differentiate symbiotically into tissue-resident macrophages (TRMs)1. However, our understanding of the origins and specialization of macrophages in human embryos is limited. Here we isolated CD45+ haematopoietic cells from human embryos at Carnegie stages 11 to 23 and subjected them to transcriptomic profiling by single-cell RNA sequencing, followed by functional characterization of a population of CD45+CD34+CD44+ yolk sac-derived myeloid-biased progenitors (YSMPs) by single-cell culture. We also mapped macrophage heterogeneity across multiple anatomical sites and identified diverse subsets, including various types of embryonic TRM (in the head, liver, lung and skin). We further traced the specification trajectories of TRMs from either yolk sac-derived primitive macrophages or YSMP-derived embryonic liver monocytes using both transcriptomic and developmental staging information, with a focus on microglia. Finally, we evaluated the molecular similarities between embryonic TRMs and their adult counterparts. Our data represent a comprehensive characterization of the spatiotemporal dynamics of early macrophage development during human embryogenesis, providing a reference for future studies of the development and function of human TRMs.


Assuntos
Macrófagos/citologia , Análise de Célula Única , Linhagem da Célula , Embrião de Mamíferos/citologia , Cabeça , Hematopoese , Humanos , Antígenos Comuns de Leucócito/metabolismo , Fígado/citologia , Fígado/embriologia , Pulmão/citologia , Macrófagos/metabolismo , Microglia/citologia , Células Progenitoras Mieloides/citologia , RNA-Seq , Pele/citologia , Análise Espaço-Temporal , Transcriptoma , Saco Vitelino/citologia
14.
J Vis Exp ; (160)2020 06 14.
Artigo em Inglês | MEDLINE | ID: mdl-32597854

RESUMO

In utero electroporation is an in vivo DNA transfer technique extensively used to study the molecular and cellular mechanisms underlying mammalian corticogenesis. This procedure takes advantage of the brain ventricles to allow the introduction of DNA of interest and uses a pair of electrodes to direct the entrance of the genetic material into the cells lining the ventricle, the neural stem cells. This method allows researchers to label the desired cells and/or manipulate the expression of genes of interest in those cells. It has multiple applications, including assays targeting neuronal migration, lineage tracing, and axonal pathfinding. An important feature of this method is its temporal and regional control, allowing circumvention of potential problems related with embryonic lethality or the lack of specific CRE driver mice. Another relevant aspect of this technique is that it helps to considerably reduce the economic and temporal limitations that involve the generation of new mouse lines, which become particularly important in the study of interactions between cell types that originate in distant areas of the brain at different developmental ages. Here we describe a double electroporation strategy that enables targeting of cell populations that are spatially and temporally separated. With this approach we can label different subtypes of cells in different locations with selected fluorescent proteins to visualize them, and/or we can manipulate genes of interest expressed by these different cells at the appropriate times. This strategy enhances the potential of in utero electroporation and provides a powerful tool to study the behavior of temporally and spatially separated cell populations that migrate to establish close contacts, as well as long-range interactions through axonal projections, reducing temporal and economic costs.


Assuntos
Encéfalo/metabolismo , DNA/administração & dosagem , Eletroporação/métodos , Embrião de Mamíferos/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Células-Tronco Neurais/metabolismo , Análise Espaço-Temporal , Animais , Encéfalo/citologia , DNA/genética , DNA/metabolismo , Embrião de Mamíferos/citologia , Feminino , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Células-Tronco Neurais/citologia , Neurogênese , Plasmídeos/administração & dosagem , Gravidez
15.
J Vis Exp ; (160)2020 06 12.
Artigo em Inglês | MEDLINE | ID: mdl-32597868

RESUMO

Human implantation, the apposition and adhesion to the uterine surface epithelia and subsequent invasion of the blastocyst into the maternal decidua, is a critical yet enigmatic biological event that has been historically difficult to study due to technical and ethical limitations. Implantation is initiated by the development of the trophectoderm to early trophoblast and subsequent differentiation into distinct trophoblast sublineages. Aberrant early trophoblast differentiation may lead to implantation failure, placental pathologies, fetal abnormalities, and miscarriage. Recently, methods have been developed to allow human embryos to grow until day 13 post-fertilization in vitro in the absence of maternal tissues, a time-period that encompasses the implantation period in humans. This has given researchers the opportunity to investigate human implantation and recapitulate the dynamics of trophoblast differentiation during this critical period without confounding maternal influences and avoiding inherent obstacles to study early embryo differentiation events in vivo. To characterize different trophoblast sublineages during implantation, we have adopted existing two-dimensional (2D) extended culture methods and developed a procedure to enzymatically digest and isolate different types of trophoblast cells for downstream assays. Embryos cultured in 2D conditions have a relatively flattened morphology and may be suboptimal in modeling in vivo three-dimensional (3D) embryonic architectures. However, trophoblast differentiation seems to be less affected as demonstrated by anticipated morphology and gene expression changes over the course of extended culture. Different trophoblast sublineages, including cytotrophoblast, syncytiotrophoblast and migratory trophoblast can be separated by size, location, and temporal emergence, and used for further characterization or experimentation. Investigation of these early trophoblast cells may be instrumental in understanding human implantation, treating common placental pathologies, and mitigating the incidence of pregnancy loss.


Assuntos
Separação Celular/métodos , Implantação do Embrião , Embrião de Mamíferos/citologia , Trofoblastos/citologia , Animais , Biomarcadores/metabolismo , Blastocisto/citologia , Forma Celular , Células Cultivadas , Gonadotropina Coriônica/farmacologia , Feminino , Humanos , Gravidez , Imagem com Lapso de Tempo , Fixação de Tecidos , Tripsina/metabolismo , Vitrificação
16.
Nat Commun ; 11(1): 3252, 2020 06 26.
Artigo em Inglês | MEDLINE | ID: mdl-32591534

RESUMO

MiDAC is one of seven distinct, large multi-protein complexes that recruit class I histone deacetylases to the genome to regulate gene expression. Despite implications of involvement in cell cycle regulation and in several cancers, surprisingly little is known about the function or structure of MiDAC. Here we show that MiDAC is important for chromosome alignment during mitosis in cancer cell lines. Mice lacking the MiDAC proteins, DNTTIP1 or MIDEAS, die with identical phenotypes during late embryogenesis due to perturbations in gene expression that result in heart malformation and haematopoietic failure. This suggests that MiDAC has an essential and unique function that cannot be compensated by other HDAC complexes. Consistent with this, the cryoEM structure of MiDAC reveals a unique and distinctive mode of assembly. Four copies of HDAC1 are positioned at the periphery with outward-facing active sites suggesting that the complex may target multiple nucleosomes implying a processive deacetylase function.


Assuntos
Desenvolvimento Embrionário , Histona Desacetilases/metabolismo , Complexos Multiproteicos/metabolismo , Sequência de Aminoácidos , Animais , Linhagem Celular , Cromatina/metabolismo , Cromossomos de Mamíferos/metabolismo , Embrião de Mamíferos/citologia , Fibroblastos/metabolismo , Redes Reguladoras de Genes , Heterozigoto , Homozigoto , Humanos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Mitose , Modelos Moleculares , Complexos Multiproteicos/química , Complexos Multiproteicos/ultraestrutura , Proteínas Nucleares/metabolismo , Domínios Proteicos , Multimerização Proteica
17.
Proc Natl Acad Sci U S A ; 117(22): 12182-12191, 2020 06 02.
Artigo em Inglês | MEDLINE | ID: mdl-32414917

RESUMO

In multicellular organisms, paralogs from gene duplication survive purifying selection by evolving tissue-specific expression and function. Whether this genetic redundancy is also selected for within a single cell type is unclear for multimember paralogs, as exemplified by the four obligatory Lef/Tcf transcription factors of canonical Wnt signaling, mainly due to the complex genetics involved. Using the developing mouse lung as a model system, we generate two quadruple conditional knockouts, four triple mutants, and various combinations of double mutants, showing that the four Lef/Tcf genes function redundantly in the presence of at least two Lef/Tcf paralogs, but additively upon losing additional paralogs to specify and maintain lung epithelial progenitors. Prelung-specification, pan-epithelial double knockouts have no lung phenotype; triple knockouts have varying phenotypes, including defective branching and tracheoesophageal fistulas; and the quadruple knockout barely forms a lung, resembling the Ctnnb1 mutant. Postlung-specification deletion of all four Lef/Tcf genes leads to branching defects, down-regulation of progenitor genes, premature alveolar differentiation, and derepression of gastrointestinal genes, again phenocopying the corresponding Ctnnb1 mutant. Our study supports a monotonic, positive signaling relationship between CTNNB1 and Lef/Tcf in lung epithelial progenitors as opposed to reported repressor functions of Lef/Tcf, and represents a thorough in vivo analysis of cell-type-specific genetic redundancy among the four Lef/Tcf paralogs.


Assuntos
Embrião de Mamíferos/metabolismo , Células-Tronco Embrionárias/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Pulmão/metabolismo , Fator 1 de Ligação ao Facilitador Linfoide/fisiologia , Células-Tronco/metabolismo , beta Catenina/metabolismo , Animais , Diferenciação Celular , Embrião de Mamíferos/citologia , Células-Tronco Embrionárias/citologia , Feminino , Fator 1-alfa Nuclear de Hepatócito/fisiologia , Pulmão/citologia , Camundongos , Camundongos Knockout , Análise de Célula Única , Células-Tronco/citologia , Proteína 1 Semelhante ao Fator 7 de Transcrição/fisiologia , Proteína 2 Semelhante ao Fator 7 de Transcrição/fisiologia , Proteínas Wnt/genética , Proteínas Wnt/metabolismo , beta Catenina/genética
18.
Nature ; 581(7806): 77-82, 2020 05.
Artigo em Inglês | MEDLINE | ID: mdl-32376949

RESUMO

Grafts of spinal-cord-derived neural progenitor cells (NPCs) enable the robust regeneration of corticospinal axons and restore forelimb function after spinal cord injury1; however, the molecular mechanisms that underlie this regeneration are unknown. Here we perform translational profiling specifically of corticospinal tract (CST) motor neurons in mice, to identify their 'regenerative transcriptome' after spinal cord injury and NPC grafting. Notably, both injury alone and injury combined with NPC grafts elicit virtually identical early transcriptomic responses in host CST neurons. However, in mice with injury alone this regenerative transcriptome is downregulated after two weeks, whereas in NPC-grafted mice this transcriptome is sustained. The regenerative transcriptome represents a reversion to an embryonic transcriptional state of the CST neuron. The huntingtin gene (Htt) is a central hub in the regeneration transcriptome; deletion of Htt significantly attenuates regeneration, which shows that Htt has a key role in neural plasticity after injury.


Assuntos
Proliferação de Células/genética , Embrião de Mamíferos/citologia , Embrião de Mamíferos/metabolismo , Regeneração Nervosa/genética , Células-Tronco Neurais/citologia , Neurônios/metabolismo , Neurônios/patologia , Transcrição Genética , Animais , Axônios/patologia , Axônios/fisiologia , Modelos Animais de Doenças , Feminino , Perfilação da Expressão Gênica , Proteína Huntingtina/genética , Camundongos , Células-Tronco Neurais/transplante , Plasticidade Neuronal , Neurônios/citologia , Neurônios/transplante , Biossíntese de Proteínas , Tratos Piramidais/citologia , Tratos Piramidais/metabolismo , Tratos Piramidais/patologia , RNA-Seq , Traumatismos da Medula Espinal/genética , Traumatismos da Medula Espinal/patologia , Transcriptoma
19.
Nat Cell Biol ; 22(6): 740-750, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-32393889

RESUMO

Cytidine base editors are powerful genetic tools that catalyse cytidine to thymidine conversion at specific genomic loci, and further improvement of the editing range and efficiency is critical for their broader applications. Through insertion of a non-sequence-specific single-stranded DNA-binding domain from Rad51 protein between Cas9 nickase and the deaminases, serial hyper cytidine base editors were generated with substantially increased activity and an expanded editing window towards the protospacer adjacent motif in both cell lines and mouse embryos. Additionally, hyeA3A-BE4max selectively catalysed cytidine conversion in TC motifs with a broader editing range and much higher activity (up to 257-fold) compared with eA3A-BE4max. Moreover, hyeA3A-BE4max specifically generated a C-to-T conversion without inducing bystander mutations in the haemoglobin gamma gene promoter to mimic a naturally occurring genetic variant for amelioration of ß-haemoglobinopathy, suggesting the therapeutic potential of the improved base editors.


Assuntos
Sistemas CRISPR-Cas , Citidina/genética , Proteínas de Ligação a DNA/metabolismo , Edição de Genes , Mutação , Rad51 Recombinase/metabolismo , Animais , Diferenciação Celular , Citidina/química , Proteínas de Ligação a DNA/genética , Embrião de Mamíferos/citologia , Embrião de Mamíferos/metabolismo , Feminino , Células HEK293 , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos ICR , Domínios Proteicos , Rad51 Recombinase/genética
20.
Nat Commun ; 11(1): 2366, 2020 05 12.
Artigo em Inglês | MEDLINE | ID: mdl-32398639

RESUMO

Epithelial bending is a fundamental process that shapes organs during development. Previously known mechanisms involve cells locally changing shape from columnar to wedge-shaped. Here we report a different mechanism that occurs without cell wedging. In mammalian salivary glands and teeth, we show that initial invagination occurs through coordinated vertical cell movement: cells towards the periphery of the placode move vertically upwards while their more central neighbours move downwards. Movement is achieved by active cell-on-cell migration: outer cells migrate with apical, centripetally polarised leading edge protrusions but remain attached to the basal lamina, depressing more central neighbours to "telescope" the epithelium downwards into underlying mesenchyme. Inhibiting protrusion formation by Arp2/3 protein blocks invagination. FGF and Hedgehog morphogen signals are required, with FGF providing a directional cue. These findings show that epithelial bending can be achieved by a morphogenetic mechanism of coordinated cell rearrangement quite distinct from previously recognised invagination processes.


Assuntos
Movimento Celular/fisiologia , Desenvolvimento Embrionário/fisiologia , Epitélio/embriologia , Dente Molar/embriologia , Glândulas Salivares/embriologia , Animais , Ectoderma/citologia , Ectoderma/embriologia , Embrião de Mamíferos/citologia , Células Epiteliais/fisiologia , Feminino , Microscopia Intravital , Masculino , Camundongos , Dente Molar/citologia , Glândulas Salivares/citologia , Técnicas de Cultura de Tecidos
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