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1.
Development ; 148(6)2021 03 29.
Artigo em Inglês | MEDLINE | ID: mdl-33782043

RESUMO

Rostro-caudal patterning of vertebrates depends on the temporally progressive activation of HOX genes within axial stem cells that fuel axial embryo elongation. Whether the pace of sequential activation of HOX genes, the 'HOX clock', is controlled by intrinsic chromatin-based timing mechanisms or by temporal changes in extrinsic cues remains unclear. Here, we studied HOX clock pacing in human pluripotent stem cell-derived axial progenitors differentiating into diverse spinal cord motor neuron subtypes. We show that the progressive activation of caudal HOX genes is controlled by a dynamic increase in FGF signaling. Blocking the FGF pathway stalled induction of HOX genes, while a precocious increase of FGF, alone or with GDF11 ligand, accelerated the HOX clock. Cells differentiated under accelerated HOX induction generated appropriate posterior motor neuron subtypes found along the human embryonic spinal cord. The pacing of the HOX clock is thus dynamically regulated by exposure to secreted cues. Its manipulation by extrinsic factors provides synchronized access to multiple human neuronal subtypes of distinct rostro-caudal identities for basic and translational applications.This article has an associated 'The people behind the papers' interview.


Assuntos
Relógios Circadianos , Proteínas de Homeodomínio/metabolismo , Neurônios Motores/metabolismo , Células-Tronco Pluripotentes/metabolismo , Benzamidas/farmacologia , Proteínas Morfogenéticas Ósseas/genética , Proteínas Morfogenéticas Ósseas/metabolismo , Proteínas Morfogenéticas Ósseas/farmacologia , Diferenciação Celular , Relógios Circadianos/efeitos dos fármacos , Difenilamina/análogos & derivados , Difenilamina/farmacologia , Embrião de Mamíferos/citologia , Embrião de Mamíferos/metabolismo , Desenvolvimento Embrionário , Fatores de Crescimento de Fibroblastos/antagonistas & inibidores , Fatores de Crescimento de Fibroblastos/metabolismo , Fatores de Crescimento de Fibroblastos/farmacologia , Regulação da Expressão Gênica no Desenvolvimento , Fatores de Diferenciação de Crescimento/genética , Fatores de Diferenciação de Crescimento/metabolismo , Fatores de Diferenciação de Crescimento/farmacologia , Proteínas de Homeodomínio/genética , Humanos , Neurônios Motores/citologia , Células-Tronco Pluripotentes/citologia , Pirimidinas/farmacologia , Transdução de Sinais/efeitos dos fármacos , Medula Espinal/metabolismo
2.
Neuron ; 107(4): 617-630.e6, 2020 08 19.
Artigo em Inglês | MEDLINE | ID: mdl-32559415

RESUMO

Stable genomic integration of exogenous transgenes is essential in neurodevelopmental and stem cell studies. Despite tools driving increasingly efficient genomic insertion with DNA vectors, transgenesis remains fundamentally hindered by the impossibility of distinguishing integrated from episomal transgenes. Here, we introduce an integration-coupled On genetic switch, iOn, which triggers gene expression upon incorporation into the host genome through transposition, thus enabling rapid and accurate identification of integration events following transfection with naked plasmids. In vitro, iOn permits rapid drug-free stable transgenesis of mouse and human pluripotent stem cells with multiple vectors. In vivo, we demonstrate faithful cell lineage tracing, assessment of regulatory elements, and mosaic analysis of gene function in somatic transgenesis experiments that reveal neural progenitor potentialities and interaction. These results establish iOn as a universally applicable strategy to accelerate and simplify genetic engineering in cultured systems and model organisms by conditioning transgene activation to genomic integration.


Assuntos
Expressão Gênica , Técnicas de Transferência de Genes , Células-Tronco Neurais , Transgenes , Animais , Linhagem da Célula , Vetores Genéticos , Humanos , Camundongos
3.
Development ; 146(14)2019 07 25.
Artigo em Inglês | MEDLINE | ID: mdl-31239243

RESUMO

Bone morphogenetic proteins (BMPs) are secreted regulators of cell fate in several developing tissues. In the embryonic spinal cord, they control the emergence of the neural crest, roof plate and distinct subsets of dorsal interneurons. Although a gradient of BMP activity has been proposed to determine cell type identity in vivo, whether this is sufficient for pattern formation in vitro is unclear. Here, we demonstrate that exposure to BMP4 initiates distinct spatial dynamics of BMP signalling within the self-emerging epithelia of both mouse and human pluripotent stem cell-derived spinal organoids. The pattern of BMP signalling results in the stereotyped spatial arrangement of dorsal neural tube cell types, and concentration, timing and duration of BMP4 exposure modulate these patterns. Moreover, differences in the duration of competence time-windows between mouse and human account for the species-specific tempo of neural differentiation. Together, this study describes efficient methods for generating patterned subsets of dorsal interneurons in spinal organoids and supports the conclusion that graded BMP activity orchestrates the spatial organization of the dorsal neural tube cellular diversity in mouse and human.


Assuntos
Proteína Morfogenética Óssea 4/fisiologia , Diferenciação Celular/genética , Organoides/fisiologia , Proteínas Smad/metabolismo , Coluna Vertebral/citologia , Animais , Linhagem da Célula/genética , Células Cultivadas , Embrião de Mamíferos , Regulação da Expressão Gênica no Desenvolvimento , Humanos , Células-Tronco Pluripotentes Induzidas/citologia , Células-Tronco Pluripotentes Induzidas/fisiologia , Interneurônios/citologia , Interneurônios/fisiologia , Camundongos , Crista Neural/citologia , Crista Neural/fisiologia , Tubo Neural/citologia , Tubo Neural/embriologia , Neurônios/citologia , Neurônios/fisiologia , Organoides/citologia , Transdução de Sinais/genética , Proteínas Smad/genética
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