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1.
Cell ; 175(3): 859-876.e33, 2018 10 18.
Artigo em Inglês | MEDLINE | ID: mdl-30318151

RESUMO

The mouse embryo has long been central to the study of mammalian development; however, elucidating the cell behaviors governing gastrulation and the formation of tissues and organs remains a fundamental challenge. A major obstacle is the lack of live imaging and image analysis technologies capable of systematically following cellular dynamics across the developing embryo. We developed a light-sheet microscope that adapts itself to the dramatic changes in size, shape, and optical properties of the post-implantation mouse embryo and captures its development from gastrulation to early organogenesis at the cellular level. We furthermore developed a computational framework for reconstructing long-term cell tracks, cell divisions, dynamic fate maps, and maps of tissue morphogenesis across the entire embryo. By jointly analyzing cellular dynamics in multiple embryos registered in space and time, we built a dynamic atlas of post-implantation mouse development that, together with our microscopy and computational methods, is provided as a resource. VIDEO ABSTRACT.


Assuntos
Linhagem da Célula , Gastrulação , Organogênese , Análise de Célula Única/métodos , Animais , Camundongos , Camundongos Endogâmicos C57BL , Modelos Estatísticos , Imagem Óptica/métodos
2.
Dev Biol ; 448(2): 71-87, 2019 04 15.
Artigo em Inglês | MEDLINE | ID: mdl-30661644

RESUMO

Ascidian species of the Phallusia and Ciona genera are distantly related, their last common ancestor dating several hundred million years ago. Although their genome sequences have extensively diverged since this radiation, Phallusia and Ciona species share almost identical early morphogenesis and stereotyped cell lineages. Here, we explored the evolution of transcriptional control between P. mammillata and C. robusta. We combined genome-wide mapping of open chromatin regions in both species with a comparative analysis of the regulatory sequences of a test set of 10 pairs of orthologous early regulatory genes with conserved expression patterns. We find that ascidian chromatin accessibility landscapes obey similar rules as in other metazoa. Open-chromatin regions are short, highly conserved within each genus and cluster around regulatory genes. The dynamics of chromatin accessibility and closest-gene expression are strongly correlated during early embryogenesis. Open-chromatin regions are highly enriched in cis-regulatory elements: 73% of 49 open chromatin regions around our test genes behaved as either distal enhancers or proximal enhancer/promoters following electroporation in Phallusia eggs. Analysis of this datasets suggests a pervasive use in ascidians of "shadow" enhancers with partially overlapping activities. Cross-species electroporations point to a deep conservation of both the trans-regulatory logic between these distantly-related ascidians and the cis-regulatory activities of individual enhancers. Finally, we found that the relative order and approximate distance to the transcription start site of open chromatin regions can be conserved between Ciona and Phallusia species despite extensive sequence divergence, a property that can be used to identify orthologous enhancers, whose regulatory activity can partially diverge.


Assuntos
Ciona/embriologia , Ciona/genética , Embrião não Mamífero/metabolismo , Evolução Molecular , Variação Genética , Sequências Reguladoras de Ácido Nucleico/genética , Urocordados/embriologia , Urocordados/genética , Animais , Sequência de Bases , Padronização Corporal/genética , Cromatina/genética , Sequência Conservada/genética , Desenvolvimento Embrionário/genética , Elementos Facilitadores Genéticos , Gástrula/embriologia , Regulação da Expressão Gênica no Desenvolvimento , Especificidade da Espécie , Fatores de Tempo
3.
EMBO Rep ; 16(3): 332-40, 2015 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-25652260

RESUMO

The accumulation of misfolded proteins in the endoplasmic reticulum (ER) activates the Unfolded Protein Response (UPR(ER)) to restore ER homeostasis. The AAA(+) ATPase p97/CDC-48 plays key roles in ER stress by promoting both ER protein degradation and transcription of UPR(ER) genes. Although the mechanisms associated with protein degradation are now well established, the molecular events involved in the regulation of gene transcription by p97/CDC-48 remain unclear. Using a reporter-based genome-wide RNAi screen in combination with quantitative proteomic analysis in Caenorhabditis elegans, we have identified RUVB-2, a AAA(+) ATPase, as a novel repressor of a subset of UPR(ER) genes. We show that degradation of RUVB-2 by CDC-48 enhances expression of ER stress response genes through an XBP1-dependent mechanism. The functional interplay between CDC-48 and RUVB-2 in controlling transcription of select UPR(ER) genes appears conserved in human cells. Together, these results describe a novel role for p97/CDC-48, whereby its role in protein degradation is integrated with its role in regulating expression of ER stress response genes.


Assuntos
Adenosina Trifosfatases/metabolismo , Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/fisiologia , Proteínas de Ciclo Celular/metabolismo , Estresse do Retículo Endoplasmático/fisiologia , Transdução de Sinais/genética , Transcrição Gênica/fisiologia , Resposta a Proteínas não Dobradas/fisiologia , Adenosina Trifosfatases/genética , Animais , Proteínas de Caenorhabditis elegans/genética , Proteínas de Ciclo Celular/genética , Estresse do Retículo Endoplasmático/genética , Proteômica/métodos , Interferência de RNA , Proteínas Repressoras/metabolismo , Proteína com Valosina
4.
Nat Biotechnol ; 41(1): 44-49, 2023 01.
Artigo em Inglês | MEDLINE | ID: mdl-36065022

RESUMO

We present a method to automatically identify and track nuclei in time-lapse microscopy recordings of entire developing embryos. The method combines deep learning and global optimization. On a mouse dataset, it reconstructs 75.8% of cell lineages spanning 1 h, as compared to 31.8% for the competing method. Our approach improves understanding of where and when cell fate decisions are made in developing embryos, tissues, and organs.


Assuntos
Blastocisto , Embrião de Mamíferos , Animais , Camundongos , Linhagem da Célula , Microscopia
5.
Nat Genet ; 55(7): 1176-1185, 2023 07.
Artigo em Inglês | MEDLINE | ID: mdl-37414952

RESUMO

Spatiotemporal orchestration of gene expression is required for proper embryonic development. The use of single-cell technologies has begun to provide improved resolution of early regulatory dynamics, including detailed molecular definitions of most cell states during mouse embryogenesis. Here we used Slide-seq to build spatial transcriptomic maps of complete embryonic day (E) 8.5 and E9.0, and partial E9.5 embryos. To support their utility, we developed sc3D, a tool for reconstructing and exploring three-dimensional 'virtual embryos', which enables the quantitative investigation of regionalized gene expression patterns. Our measurements along the main embryonic axes of the developing neural tube revealed several previously unannotated genes with distinct spatial patterns. We also characterized the conflicting transcriptional identity of 'ectopic' neural tubes that emerge in Tbx6 mutant embryos. Taken together, we present an experimental and computational framework for the spatiotemporal investigation of whole embryonic structures and mutant phenotypes.


Assuntos
Organogênese , Transcriptoma , Camundongos , Animais , Transcriptoma/genética , Organogênese/genética , Desenvolvimento Embrionário/genética , Embrião de Mamíferos , Fenótipo , Regulação da Expressão Gênica no Desenvolvimento/genética , Proteínas com Domínio T/genética
6.
Science ; 369(6500)2020 07 10.
Artigo em Inglês | MEDLINE | ID: mdl-32646972

RESUMO

Marine invertebrate ascidians display embryonic reproducibility: Their early embryonic cell lineages are considered invariant and are conserved between distantly related species, despite rapid genomic divergence. Here, we address the drivers of this reproducibility. We used light-sheet imaging and automated cell segmentation and tracking procedures to systematically quantify the behavior of individual cells every 2 minutes during Phallusia mammillata embryogenesis. Interindividual reproducibility was observed down to the area of individual cell contacts. We found tight links between the reproducibility of embryonic geometries and asymmetric cell divisions, controlled by differential sister cell inductions. We combined modeling and experimental manipulations to show that the area of contact between signaling and responding cells is a key determinant of cell communication. Our work establishes the geometric control of embryonic inductions as an alternative to classical morphogen gradients and suggests that the range of cell signaling sets the scale at which embryonic reproducibility is observed.


Assuntos
Urocordados/citologia , Urocordados/embriologia , Animais , Comunicação Celular , Divisão Celular , Rastreamento de Células , Reprodução
7.
Science ; 370(6522)2020 12 11.
Artigo em Inglês | MEDLINE | ID: mdl-33303587

RESUMO

Post-implantation embryogenesis is a highly dynamic process comprising multiple lineage decisions and morphogenetic changes that are inaccessible to deep analysis in vivo. We found that pluripotent mouse embryonic stem cells (mESCs) form aggregates that upon embedding in an extracellular matrix compound induce the formation of highly organized "trunk-like structures" (TLSs) comprising the neural tube and somites. Comparative single-cell RNA sequencing analysis confirmed that this process is highly analogous to mouse development and follows the same stepwise gene-regulatory program. Tbx6 knockout TLSs developed additional neural tubes mirroring the embryonic mutant phenotype, and chemical modulation could induce excess somite formation. TLSs thus reveal an advanced level of self-organization and provide a powerful platform for investigating post-implantation embryogenesis in a dish.


Assuntos
Desenvolvimento Embrionário/fisiologia , Células-Tronco Embrionárias Murinas/fisiologia , Tubo Neural/embriologia , Somitos/embriologia , Animais , Desenvolvimento Embrionário/genética , Regulação da Expressão Gênica no Desenvolvimento , Camundongos , Camundongos Knockout , Piridinas/farmacologia , Pirimidinas/farmacologia , Proteínas com Domínio T/genética , Proteínas Wnt/antagonistas & inibidores
8.
Elife ; 72018 03 29.
Artigo em Inglês | MEDLINE | ID: mdl-29595475

RESUMO

During development, coordinated cell behaviors orchestrate tissue and organ morphogenesis. Detailed descriptions of cell lineages and behaviors provide a powerful framework to elucidate the mechanisms of morphogenesis. To study the cellular basis of limb development, we imaged transgenic fluorescently-labeled embryos from the crustacean Parhyale hawaiensis with multi-view light-sheet microscopy at high spatiotemporal resolution over several days of embryogenesis. The cell lineage of outgrowing thoracic limbs was reconstructed at single-cell resolution with new software called Massive Multi-view Tracker (MaMuT). In silico clonal analyses suggested that the early limb primordium becomes subdivided into anterior-posterior and dorsal-ventral compartments whose boundaries intersect at the distal tip of the growing limb. Limb-bud formation is associated with spatial modulation of cell proliferation, while limb elongation is also driven by preferential orientation of cell divisions along the proximal-distal growth axis. Cellular reconstructions were predictive of the expression patterns of limb development genes including the BMP morphogen Decapentaplegic.


Assuntos
Anfípodes/embriologia , Linhagem da Célula , Biologia Computacional/métodos , Extremidades/embriologia , Processamento de Imagem Assistida por Computador/métodos , Morfogênese , Imagem Óptica/métodos , Animais , Fluorescência , Genes Reporter , Software , Análise Espaço-Temporal , Coloração e Rotulagem
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