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1.
Nat Commun ; 15(1): 7860, 2024 Sep 09.
Artículo en Inglés | MEDLINE | ID: mdl-39251590

RESUMEN

Pluripotent mouse embryonic stem cells (ESCs) can differentiate to all germ layers and serve as an in vitro model of embryonic development. To better understand the differentiation paths traversed by ESCs committing to different lineages, we track individual differentiating ESCs by timelapse imaging followed by multiplexed high-dimensional Imaging Mass Cytometry (IMC) protein quantification. This links continuous live single-cell molecular NANOG and cellular dynamics quantification over 5-6 generations to protein expression of 37 different molecular regulators in the same single cells at the observation endpoints. Using this unique data set including kinship history and live lineage marker detection, we show that NANOG downregulation occurs generations prior to, but is not sufficient for neuroectoderm marker Sox1 upregulation. We identify a developmental cell type co-expressing both the canonical Sox1 neuroectoderm and FoxA2 endoderm markers in vitro and confirm the presence of such a population in the post-implantation embryo. RNASeq reveals cells co-expressing SOX1 and FOXA2 to have a unique cell state characterized by expression of both endoderm as well as neuroectoderm genes suggesting lineage potential towards both germ layers.


Asunto(s)
Diferenciación Celular , Regulación del Desarrollo de la Expresión Génica , Factor Nuclear 3-beta del Hepatocito , Células Madre Embrionarias de Ratones , Factores de Transcripción SOXB1 , Animales , Ratones , Factor Nuclear 3-beta del Hepatocito/metabolismo , Factor Nuclear 3-beta del Hepatocito/genética , Factores de Transcripción SOXB1/metabolismo , Factores de Transcripción SOXB1/genética , Células Madre Embrionarias de Ratones/metabolismo , Células Madre Embrionarias de Ratones/citología , Rastreo Celular/métodos , Proteína Homeótica Nanog/metabolismo , Proteína Homeótica Nanog/genética , Linaje de la Célula , Endodermo/metabolismo , Endodermo/citología , Análisis de la Célula Individual/métodos , Desarrollo Embrionario/genética , Placa Neural/metabolismo , Placa Neural/embriología , Placa Neural/citología , Embrión de Mamíferos/metabolismo , Embrión de Mamíferos/citología
2.
Neuron ; 111(8): 1241-1263.e16, 2023 04 19.
Artículo en Inglés | MEDLINE | ID: mdl-36796357

RESUMEN

Cortical projection neurons polarize and form an axon while migrating radially. Even though these dynamic processes are closely interwoven, they are regulated separately-the neurons terminate their migration when reaching their destination, the cortical plate, but continue to grow their axons. Here, we show that in rodents, the centrosome distinguishes these processes. Newly developed molecular tools modulating centrosomal microtubule nucleation combined with in vivo imaging uncovered that dysregulation of centrosomal microtubule nucleation abrogated radial migration without affecting axon formation. Tightly regulated centrosomal microtubule nucleation was required for periodic formation of the cytoplasmic dilation at the leading process, which is essential for radial migration. The microtubule nucleating factor γ-tubulin decreased at neuronal centrosomes during the migratory phase. As distinct microtubule networks drive neuronal polarization and radial migration, this provides insight into how neuronal migratory defects occur without largely affecting axonal tracts in human developmental cortical dysgeneses, caused by mutations in γ-tubulin.


Asunto(s)
Neuronas , Tubulina (Proteína) , Humanos , Tubulina (Proteína)/metabolismo , Neuronas/fisiología , Axones/metabolismo , Microtúbulos/metabolismo , Centrosoma , Encéfalo/metabolismo
3.
Trends Cell Biol ; 32(8): 707-719, 2022 08.
Artículo en Inglés | MEDLINE | ID: mdl-35750615

RESUMEN

Stem cells are at the source of creating cellular diversity. Multiple mechanisms, including basic cell biological processes, regulate their fate. The centrosome is at the core of many stem cell functions and recent work highlights the association of distinct proteins at the centrosome in stem cell differentiation. As showcased by a novel centrosome protein regulating neural stem cell differentiation, it is timely to review the heterogeneity of the centrosome at protein and RNA levels and how this impacts their function in stem and progenitor cells. Together with evidence for heterogeneity of other organelles so far considered as similar between cells, we call for exploring the cell type-specific composition of organelles as a way to expand protein function in development with relevance to regenerative medicine.


Asunto(s)
Centrosoma , Células-Madre Neurales , Diferenciación Celular/fisiología , Centrosoma/metabolismo , Humanos , Sistema Nervioso , Orgánulos
4.
J Exp Med ; 219(1)2022 01 03.
Artículo en Inglés | MEDLINE | ID: mdl-34817548

RESUMEN

Transcription factors (TFs) regulate cell fates, and their expression must be tightly regulated. Autoregulation is assumed to regulate many TFs' own expression to control cell fates. Here, we manipulate and quantify the (auto)regulation of PU.1, a TF controlling hematopoietic stem and progenitor cells (HSPCs), and correlate it to their future fates. We generate transgenic mice allowing both inducible activation of PU.1 and noninvasive quantification of endogenous PU.1 protein expression. The quantified HSPC PU.1 dynamics show that PU.1 up-regulation occurs as a consequence of hematopoietic differentiation independently of direct fast autoregulation. In contrast, inflammatory signaling induces fast PU.1 up-regulation, which does not require PU.1 expression or its binding to its own autoregulatory enhancer. However, the increased PU.1 levels induced by inflammatory signaling cannot be sustained via autoregulation after removal of the signaling stimulus. We conclude that PU.1 overexpression induces HSC differentiation before PU.1 up-regulation, only later generating cell types with intrinsically higher PU.1.


Asunto(s)
Diferenciación Celular/genética , Células Madre Hematopoyéticas/metabolismo , Homeostasis/genética , Proteínas Proto-Oncogénicas/genética , Transactivadores/genética , Regulación hacia Arriba/genética , Animales , Células Cultivadas , Expresión Génica , Masculino , Ratones Endogámicos C57BL , Ratones Transgénicos , Microscopía Fluorescente/métodos , Proteínas Proto-Oncogénicas/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transducción de Señal/genética , Imagen de Lapso de Tiempo/métodos , Transactivadores/metabolismo
5.
Nature ; 567(7746): 113-117, 2019 03.
Artículo en Inglés | MEDLINE | ID: mdl-30787442

RESUMEN

The expansion of brain size is accompanied by a relative enlargement of the subventricular zone during development. Epithelial-like neural stem cells divide in the ventricular zone at the ventricles of the embryonic brain, self-renew and generate basal progenitors1 that delaminate and settle in the subventricular zone in enlarged brain regions2. The length of time that cells stay in the subventricular zone is essential for controlling further amplification and fate determination. Here we show that the interphase centrosome protein AKNA has a key role in this process. AKNA localizes at the subdistal appendages of the mother centriole in specific subtypes of neural stem cells, and in almost all basal progenitors. This protein is necessary and sufficient to organize centrosomal microtubules, and promote their nucleation and growth. These features of AKNA are important for mediating the delamination process in the formation of the subventricular zone. Moreover, AKNA regulates the exit from the subventricular zone, which reveals the pivotal role of centrosomal microtubule organization in enabling cells to both enter and remain in the subventricular zone. The epithelial-to-mesenchymal transition is also regulated by AKNA in other epithelial cells, demonstrating its general importance for the control of cell delamination.


Asunto(s)
Centrosoma/metabolismo , Proteínas de Unión al ADN/metabolismo , Ventrículos Laterales/citología , Ventrículos Laterales/embriología , Microtúbulos/metabolismo , Neurogénesis , Proteínas Nucleares/metabolismo , Factores de Transcripción/metabolismo , Animales , Movimiento Celular , Células Cultivadas , Células Epiteliales/metabolismo , Transición Epitelial-Mesenquimal , Humanos , Uniones Intercelulares/metabolismo , Interfase , Ventrículos Laterales/anatomía & histología , Glándulas Mamarias Animales/citología , Ratones , Tamaño de los Órganos , Organoides/citología
6.
Sci Rep ; 8(1): 5101, 2018 03 23.
Artículo en Inglés | MEDLINE | ID: mdl-29572515

RESUMEN

The serotonin neurotransmitter system is widespread in the brain and implicated in modulation of neuronal responses to other neurotransmitters. Among 14 serotonin receptor subtypes, 5-HT2cR plays a pivotal role in controlling neuronal network excitability. Serotonergic activity conveyed through receptor 5-HT2cR is regulated post-transcriptionally via two mechanisms, alternative splicing and A-to-I RNA editing. Brain-specific small nucleolar RNA SNORD115 harbours a phylogenetically conserved 18-nucleotide antisense element with perfect complementarity to the region of 5ht2c primary transcript that undergoes post-transcriptional changes. Previous 5ht2c minigene studies have implicated SNORD115 in fine-tuning of both post-transcriptional events. We monitored post-transcriptional changes of endogenous 5ht2c transcripts during neuronal differentiation. Both SNORD115 and 5ht2c were upregulated upon neuronal commitment. We detected increased 5ht2c alternative exon Vb inclusion already at the stage of neuronal progenitors, and more extensive A-to-I editing of non-targeted sites A and B compared to adjacent adenosines at sites E, C and D throughout differentiation. As the extent of editing is known to positively correlate with exon Vb usage while it reduces receptor functionality, our data support the model where SNORD115 directly promotes alternative exon inclusion without the requirement for conversion of key adenosines to inosines, thereby favouring production of full-length receptor isoforms with higher potency.


Asunto(s)
Neurogénesis , Neuronas/citología , Células Madre Pluripotentes/citología , ARN Mensajero/genética , ARN Nucleolar Pequeño/genética , Receptor de Serotonina 5-HT2C/genética , Empalme Alternativo , Animales , Línea Celular , Regulación del Desarrollo de la Expresión Génica , Ratones , Neuronas/metabolismo , Células Madre Pluripotentes/metabolismo , Transcriptoma
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