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1.
Nat Commun ; 12(1): 4502, 2021 07 23.
Artigo em Inglês | MEDLINE | ID: mdl-34301937

RESUMO

Cells in many tissues, such as bone, muscle, and placenta, fuse into syncytia to acquire new functions and transcriptional programs. While it is known that fused cells are specialized, it is unclear whether cell-fusion itself contributes to programmatic-changes that generate the new cellular state. Here, we address this by employing a fusogen-mediated, cell-fusion system to create syncytia from undifferentiated cells. RNA-Seq analysis reveals VSV-G-induced cell fusion precedes transcriptional changes. To gain mechanistic insights, we measure the plasma membrane surface area after cell-fusion and observe it diminishes through increases in endocytosis. Consequently, glucose transporters internalize, and cytoplasmic glucose and ATP transiently decrease. This reduced energetic state activates AMPK, which inhibits YAP1, causing transcriptional-reprogramming and cell-cycle arrest. Impairing either endocytosis or AMPK activity prevents YAP1 inhibition and cell-cycle arrest after fusion. Together, these data demonstrate plasma membrane diminishment upon cell-fusion causes transient nutrient stress that may promote transcriptional-reprogramming independent from extrinsic cues.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Membrana Celular/metabolismo , Núcleo Celular/metabolismo , Glicoproteínas de Membrana/metabolismo , Fatores de Transcrição/metabolismo , Transcrição Genética/genética , Proteínas do Envelope Viral/metabolismo , Proteínas Quinases Ativadas por AMP/genética , Proteínas Quinases Ativadas por AMP/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Animais , Transporte Biológico , Fusão Celular , Linhagem Celular , Linhagem Celular Tumoral , Células Cultivadas , Células Gigantes/metabolismo , Células HEK293 , Humanos , Glicoproteínas de Membrana/genética , Camundongos , RNA-Seq/métodos , Transdução de Sinais/genética , Fatores de Transcrição/genética , Proteínas do Envelope Viral/genética
2.
Prog Neurobiol ; 199: 101966, 2021 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-33249090

RESUMO

Reconstructing the genealogy of every cell that makes up an organism remains a long-standing challenge in developmental biology. Besides its relevance for understanding the mechanisms underlying normal and pathological development, resolving the lineage origin of cell types will be crucial to create these types on-demand. Multiple strategies have been deployed towards the problem of lineage tracing, ranging from direct observation to sophisticated genetic approaches. Here we discuss the achievements and limitations of past and current technology. Finally, we speculate about the future of lineage tracing and how to reach the next milestones in the field.

3.
Nat Neurosci ; 23(12): 1618-1628, 2020 12.
Artigo em Inglês | MEDLINE | ID: mdl-32719561

RESUMO

We present CLADES (cell lineage access driven by an edition sequence), a technology for cell lineage studies based on CRISPR-Cas9 techniques. CLADES relies on a system of genetic switches to activate and inactivate reporter genes in a predetermined order. Targeting CLADES to progenitor cells allows the progeny to inherit a sequential cascade of reporters, thereby coupling birth order to reporter expression. This system, which can also be temporally induced by heat shock, enables the temporal resolution of lineage development and can therefore be used to deconstruct an extended cell lineage by tracking the reporters expressed in the progeny. When targeted to the germ line, the same cascade progresses across animal generations, predominantly marking each generation with the corresponding combination of reporters. CLADES therefore offers an innovative strategy for making programmable cascades of genes that can be used for genetic manipulation or to record serial biological events.


Assuntos
Linhagem da Célula/genética , Animais , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Reparo do DNA , Drosophila melanogaster , Técnicas de Introdução de Genes , Genes Reporter/genética , Proteínas de Choque Térmico/genética , Células-Tronco Pluripotentes Induzidas , Edição de RNA , Ativação Transcricional , Peixe-Zebra
4.
Open Biol ; 9(12): 190229, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31822210

RESUMO

The first meeting exclusively dedicated to the 'High-throughput dense reconstruction of cell lineages' took place at Janelia Research Campus (Howard Hughes Medical Institute) from 14 to 18 April 2019. Organized by Tzumin Lee, Connie Cepko, Jorge Garcia-Marques and Isabel Espinosa-Medina, this meeting echoed the recent eruption of new tools that allow the reconstruction of lineages based on the phylogenetic analysis of DNA mutations induced during development. Combined with single-cell RNA sequencing, these tools promise to solve the lineage of complex model organisms at single-cell resolution. Here, we compile the conference consensus on the technological and computational challenges emerging from the use of the new strategies, as well as potential solutions.


Assuntos
Linhagem da Célula/genética , Rastreamento de Células , Perfilação da Expressão Gênica , Sequenciamento de Nucleotídeos em Larga Escala , Imagem Molecular , Animais , Sistemas CRISPR-Cas , Rastreamento de Células/métodos , Biologia Computacional , Código de Barras de DNA Taxonômico , Perfilação da Expressão Gênica/métodos , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Humanos , Imagem Molecular/métodos , Mutação , Filogenia , Análise de Célula Única/métodos
5.
Neuron ; 104(2): 227-238.e7, 2019 10 23.
Artigo em Inglês | MEDLINE | ID: mdl-31395429

RESUMO

Gaining independent genetic access to discrete cell types is critical to interrogate their biological functions as well as to deliver precise gene therapy. Transcriptomics has allowed us to profile cell populations with extraordinary precision, revealing that cell types are typically defined by a unique combination of genetic markers. Given the lack of adequate tools to target cell types based on multiple markers, most cell types remain inaccessible to genetic manipulation. Here we present CaSSA, a platform to create unlimited genetic switches based on CRISPR/Cas9 (Ca) and the DNA repair mechanism known as single-strand annealing (SSA). CaSSA allows engineering of independent genetic switches, each responding to a specific gRNA. Expressing multiple gRNAs in specific patterns enables multiplex cell-type-specific manipulations and combinatorial genetic targeting. CaSSA is a new genetic tool that conceptually works as an unlimited number of recombinases and will facilitate genetic access to cell types in diverse organisms.


Assuntos
Sistemas CRISPR-Cas , Reparo do DNA , Marcação de Genes/métodos , Animais , Drosophila , Técnicas Genéticas , RNA Guia , Recombinases/genética , Peixe-Zebra
6.
Clin Auton Res ; 28(1): 13-21, 2018 02.
Artigo em Inglês | MEDLINE | ID: mdl-29103139

RESUMO

We recently defined genetic traits that distinguish sympathetic from parasympathetic neurons, both preganglionic and ganglionic (Espinosa-Medina et al., Science 354:893-897, 2016). By this set of criteria, we found that the sacral autonomic outflow is sympathetic, not parasympathetic as has been thought for more than a century. Proposing such a belated shift in perspective begs the question why the new criterion (cell types defined by their genetic make-up and dependencies) should be favored over the anatomical, physiological and pharmacological considerations of long ago that inspired the "parasympathetic" classification. After a brief reminder of the former, we expound the weaknesses of the latter and argue that the novel genetic definition helps integrating neglected anatomical and physiological observations and clearing the path for future research.


Assuntos
Gânglios Parassimpáticos/anatomia & histologia , Gânglios Simpáticos/anatomia & histologia , Região Sacrococcígea/anatomia & histologia , Medula Espinal/anatomia & histologia , Humanos
7.
Proc Natl Acad Sci U S A ; 114(45): 11980-11985, 2017 11 07.
Artigo em Inglês | MEDLINE | ID: mdl-29078343

RESUMO

Most of the enteric nervous system derives from the "vagal" neural crest, lying at the level of somites 1-7, which invades the digestive tract rostro-caudally from the foregut to the hindgut. Little is known about the initial phase of this colonization, which brings enteric precursors into the foregut. Here we show that the "vagal crest" subsumes two populations of enteric precursors with contrasted origins, initial modes of migration, and destinations. Crest cells adjacent to somites 1 and 2 produce Schwann cell precursors that colonize the vagus nerve, which in turn guides them into the esophagus and stomach. Crest cells adjacent to somites 3-7 belong to the crest streams contributing to sympathetic chains: they migrate ventrally, seed the sympathetic chains, and colonize the entire digestive tract thence. Accordingly, enteric ganglia, like sympathetic ones, are atrophic when deprived of signaling through the tyrosine kinase receptor ErbB3, while half of the esophageal ganglia require, like parasympathetic ones, the nerve-associated form of the ErbB3 ligand, Neuregulin-1. These dependencies might bear relevance to Hirschsprung disease, with which alleles of Neuregulin-1 are associated.


Assuntos
Sistema Nervoso Entérico/citologia , Gânglios Simpáticos/citologia , Trato Gastrointestinal/embriologia , Crista Neural/citologia , Neuregulina-1/genética , Receptor ErbB-3/genética , Células de Schwann/citologia , Animais , Embrião de Galinha , Trato Gastrointestinal/inervação , Doença de Hirschsprung/genética , Camundongos , Neuregulina-1/metabolismo , Neurogênese/genética , Neurogênese/fisiologia , Receptor ErbB-3/metabolismo , Nervo Vago/citologia
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