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1.
EMBO J ; 2024 Jul 12.
Artículo en Inglés | MEDLINE | ID: mdl-38997504

RESUMEN

Cell communication coordinates developmental processes, maintains homeostasis, and contributes to disease. Therefore, understanding the relationship between cells in a shared environment is crucial. Here we introduce Positive Ultra-bright Fluorescent Fusion For Identifying Neighbours (PUFFFIN), a cell neighbour-labelling system based upon secretion and uptake of positively supercharged fluorescent protein s36GFP. We fused s36GFP to mNeonGreen or to a HaloTag, facilitating ultra-bright, sensitive, colour-of-choice labelling. Secretor cells transfer PUFFFIN to neighbours while retaining nuclear mCherry, making identification, isolation, and investigation of live neighbours straightforward. PUFFFIN can be delivered to cells, tissues, or embryos on a customisable single-plasmid construct composed of interchangeable components with the option to incorporate any transgene. This versatility enables the manipulation of cell properties, while simultaneously labelling surrounding cells, in cell culture or in vivo. We use PUFFFIN to ask whether pluripotent cells adjust the pace of differentiation to synchronise with their neighbours during exit from naïve pluripotency. PUFFFIN offers a simple, sensitive, customisable approach to profile non-cell-autonomous responses to natural or induced changes in cell identity or behaviour.

2.
Development ; 151(1)2024 Jan 01.
Artículo en Inglés | MEDLINE | ID: mdl-38165174

RESUMEN

Cell-cell interactions are central to development, but exploring how a change in any given cell relates to changes in the neighbour of that cell can be technically challenging. Here, we review recent developments in synthetic biology and image analysis that are helping overcome this problem. We highlight the opportunities presented by these advances and discuss opportunities and limitations in applying them to developmental model systems.


Asunto(s)
Comunicación Celular , Biología Sintética
3.
Nat Rev Mol Cell Biol ; 21(10): 568-569, 2020 10.
Artículo en Inglés | MEDLINE | ID: mdl-32820268
4.
Nature ; 583(7817): 585-589, 2020 07.
Artículo en Inglés | MEDLINE | ID: mdl-32669716

RESUMEN

Bone marrow transplantation therapy relies on the life-long regenerative capacity of haematopoietic stem cells (HSCs)1,2. HSCs present a complex variety of regenerative behaviours at the clonal level, but the mechanisms underlying this diversity are still undetermined3-11. Recent advances in single-cell RNA sequencing have revealed transcriptional differences among HSCs, providing a possible explanation for their functional heterogeneity12-17. However, the destructive nature of sequencing assays prevents simultaneous observation of stem cell state and function. To solve this challenge, we implemented expressible lentiviral barcoding, which enabled simultaneous analysis of lineages and transcriptomes from single adult HSCs and their clonal trajectories during long-term bone marrow reconstitution. Analysis of differential gene expression between clones with distinct behaviour revealed an intrinsic molecular signature that characterizes functional long-term repopulating HSCs. Probing this signature through in vivo CRISPR screening, we found the transcription factor TCF15 to be required and sufficient to drive HSC quiescence and long-term self-renewal. In situ, Tcf15 expression labels the most primitive subset of true multipotent HSCs. In conclusion, our work elucidates clone-intrinsic molecular programmes associated with functional stem cell heterogeneity and identifies a mechanism for the maintenance of the self-renewing HSC state.


Asunto(s)
Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Linaje de la Célula , Hematopoyesis , Células Madre Hematopoyéticas/citología , Células Madre Hematopoyéticas/metabolismo , Análisis de la Célula Individual , Animales , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Sistemas CRISPR-Cas , Autorrenovación de las Células , Femenino , Ratones
5.
Development ; 149(12)2022 06 15.
Artículo en Inglés | MEDLINE | ID: mdl-35616331

RESUMEN

Cell-cell interactions govern differentiation and cell competition in pluripotent cells during early development, but the investigation of such processes is hindered by a lack of efficient analysis tools. Here, we introduce SyNPL: clonal pluripotent stem cell lines that employ optimised Synthetic Notch (SynNotch) technology to report cell-cell interactions between engineered 'sender' and 'receiver' cells in cultured pluripotent cells and chimaeric mouse embryos. A modular design makes it straightforward to adapt the system for programming differentiation decisions non-cell-autonomously in receiver cells in response to direct contact with sender cells. We demonstrate the utility of this system by enforcing neuronal differentiation at the boundary between two cell populations. In summary, we provide a new adaptation of SynNotch technology that could be used to identify cell interactions and to profile changes in gene or protein expression that result from direct cell-cell contact with defined cell populations in culture and in early embryos, and that can be customised to generate synthetic patterning of cell fate decisions.


Asunto(s)
Células Madre Pluripotentes , Animales , Comunicación Celular , Diferenciación Celular/genética , Línea Celular , Células Cultivadas , Ratones
6.
Development ; 149(15)2022 08 01.
Artículo en Inglés | MEDLINE | ID: mdl-35781329

RESUMEN

Cell fate determination is a necessary and tightly regulated process for producing different cell types and structures during development. Cranial neural crest cells (CNCCs) are unique to vertebrate embryos and emerge from the neural plate borders into multiple cell lineages that differentiate into bone, cartilage, neurons and glial cells. We have previously reported that Irf6 genetically interacts with Twist1 during CNCC-derived tissue formation. Here, we have investigated the mechanistic role of Twist1 and Irf6 at early stages of craniofacial development. Our data indicate that TWIST1 is expressed in endocytic vesicles at the apical surface and interacts with ß/δ-catenins during neural tube closure, and Irf6 is involved in defining neural fold borders by restricting AP2α expression. Twist1 suppresses Irf6 and other epithelial genes in CNCCs during the epithelial-to-mesenchymal transition (EMT) process and cell migration. Conversely, a loss of Twist1 leads to a sustained expression of epithelial and cell adhesion markers in migratory CNCCs. Disruption of TWIST1 phosphorylation in vivo leads to epidermal blebbing, edema, neural tube defects and CNCC-derived structural abnormalities. Altogether, this study describes a previously uncharacterized function of mammalian Twist1 and Irf6 in the neural tube and CNCCs, and provides new target genes for Twist1 that are involved in cytoskeletal remodeling.


Asunto(s)
Cresta Neural , Tubo Neural , Animales , Cateninas , Regulación del Desarrollo de la Expresión Génica , Mamíferos/genética , Cráneo/metabolismo , Catenina delta
7.
Development ; 147(12)2020 06 22.
Artículo en Inglés | MEDLINE | ID: mdl-32487737

RESUMEN

The intrinsic mechanisms that link extracellular signalling to the onset of neural differentiation are not well understood. In pluripotent mouse cells, BMP blocks entry into the neural lineage via transcriptional upregulation of inhibitor of differentiation (Id) factors. We have previously identified the major binding partner of Id proteins in pluripotent cells as the basic helix-loop-helix (bHLH) transcription factor (TF) E2A. Id1 can prevent E2A from forming heterodimers with bHLH TFs or from forming homodimers. Here, we show that overexpression of a forced E2A homodimer is sufficient to drive robust neural commitment in pluripotent cells, even under non-permissive conditions. Conversely, we find that E2A null cells display a defect in their neural differentiation capacity. E2A acts as an upstream activator of neural lineage genes, including Sox1 and Foxd4, and as a repressor of Nodal signalling. Our results suggest a crucial role for E2A in establishing neural lineage commitment in pluripotent cells.


Asunto(s)
Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Diferenciación Celular , Neuronas/metabolismo , Regiones no Traducidas 3' , Animales , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/deficiencia , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Sistemas CRISPR-Cas/genética , Linaje de la Célula , Autorrenovación de las Células , Dimerización , Ratones , Células Madre Embrionarias de Ratones/citología , Células Madre Embrionarias de Ratones/metabolismo , Neuronas/citología , Factor 3 de Transcripción de Unión a Octámeros/deficiencia , Factor 3 de Transcripción de Unión a Octámeros/genética , Factor 3 de Transcripción de Unión a Octámeros/metabolismo , ARN Guía de Kinetoplastida/metabolismo , Factores de Transcripción SOXB1/deficiencia , Factores de Transcripción SOXB1/genética , Factores de Transcripción SOXB1/metabolismo , Transcriptoma , Regulación hacia Arriba
8.
Development ; 146(21)2019 11 08.
Artículo en Inglés | MEDLINE | ID: mdl-31601548

RESUMEN

A switch from E- to N-cadherin regulates the transition from pluripotency to neural identity, but the mechanism by which cadherins regulate differentiation was previously unknown. Here, we show that the acquisition of N-cadherin stabilises neural identity by dampening anti-neural signals. We use quantitative image analysis to show that N-cadherin promotes neural differentiation independently of its effects on cell cohesiveness. We reveal that cadherin switching diminishes the level of nuclear ß-catenin, and that N-cadherin also dampens FGF activity and consequently stabilises neural fate. Finally, we compare the timing of cadherin switching and differentiation in vivo and in vitro, and find that this process becomes dysregulated during in vitro differentiation. We propose that N-cadherin helps to propagate a stable neural identity throughout the emerging neuroepithelium, and that dysregulation of this process contributes to asynchronous differentiation in culture.


Asunto(s)
Cadherinas/fisiología , Células Madre Embrionarias/citología , Neuronas/citología , beta Catenina/fisiología , Animales , Diferenciación Celular , Linaje de la Célula , Núcleo Celular/fisiología , Células Cultivadas , Factores de Crecimiento de Fibroblastos/fisiología , Estratos Germinativos/fisiología , Ratones , Ratones Transgénicos , Células Madre Pluripotentes/citología
9.
PLoS Biol ; 17(8): e3000388, 2019 08.
Artículo en Inglés | MEDLINE | ID: mdl-31398189

RESUMEN

Methods for measuring the properties of individual cells within their native 3D environment will enable a deeper understanding of embryonic development, tissue regeneration, and tumorigenesis. However, current methods for segmenting nuclei in 3D tissues are not designed for situations in which nuclei are densely packed, nonspherical, or heterogeneous in shape, size, or texture, all of which are true of many embryonic and adult tissue types as well as in many cases for cells differentiating in culture. Here, we overcome this bottleneck by devising a novel method based on labelling the nuclear envelope (NE) and automatically distinguishing individual nuclei using a tree-structured ridge-tracing method followed by shape ranking according to a trained classifier. The method is fast and makes it possible to process images that are larger than the computer's memory. We consistently obtain accurate segmentation rates of >90%, even for challenging images such as mid-gestation embryos or 3D cultures. We provide a 3D editor and inspector for the manual curation of the segmentation results as well as a program to assess the accuracy of the segmentation. We have also generated a live reporter of the NE that can be used to track live cells in 3 dimensions over time. We use this to monitor the history of cell interactions and occurrences of neighbour exchange within cultures of pluripotent cells during differentiation. We provide these tools in an open-access user-friendly format.


Asunto(s)
Procesamiento de Imagen Asistido por Computador/métodos , Imagenología Tridimensional/métodos , Reconocimiento de Normas Patrones Automatizadas/métodos , Algoritmos , Animales , Núcleo Celular/fisiología , Colorantes Fluorescentes , Humanos , Indoles , Lamina Tipo B , Membrana Nuclear/metabolismo , Membrana Nuclear/fisiología
10.
Cell Mol Life Sci ; 78(9): 4435-4450, 2021 May.
Artículo en Inglés | MEDLINE | ID: mdl-33796894

RESUMEN

During early neural development, changes in signalling inform the expression of transcription factors that in turn instruct changes in cell identity. At the same time, switches in adhesion molecule expression result in cellular rearrangements that define the morphology of the emerging neural tube. It is becoming increasingly clear that these two processes influence each other; adhesion molecules do not simply operate downstream of or in parallel with changes in cell identity but rather actively feed into cell fate decisions. Why are differentiation and adhesion so tightly linked? It is now over 60 years since Conrad Waddington noted the remarkable "Constancy of the Wild Type" (Waddington in Nature 183: 1654-1655, 1959) yet we still do not fully understand the mechanisms that make development so reproducible. Conversely, we do not understand why directed differentiation of cells in a dish is sometimes unpredictable and difficult to control. It has long been suggested that cells make decisions as 'local cooperatives' rather than as individuals (Gurdon in Nature 336: 772-774, 1988; Lander in Cell 144: 955-969, 2011). Given that the cadherin family of adhesion molecules can simultaneously influence morphogenesis and signalling, it is tempting to speculate that they may help coordinate cell fate decisions between neighbouring cells in the embryo to ensure fidelity of patterning, and that the uncoupling of these processes in a culture dish might underlie some of the problems with controlling cell fate decisions ex-vivo. Here we review the expression and function of cadherins during early neural development and discuss how and why they might modulate signalling and differentiation as neural tissues are formed.


Asunto(s)
Cadherinas/metabolismo , Cresta Neural/metabolismo , Animales , Cadherinas/química , Cadherinas/clasificación , Adhesión Celular , Diferenciación Celular , Evolución Molecular , Humanos , Cresta Neural/citología , Cresta Neural/crecimiento & desarrollo , Neurogénesis , Transducción de Señal , Factores de Transcripción/genética , Factores de Transcripción/metabolismo
11.
Development ; 145(18)2018 09 21.
Artículo en Inglés | MEDLINE | ID: mdl-30115626

RESUMEN

Diffusible signals are known to orchestrate patterning during embryogenesis, yet diffusion is sensitive to noise. The fact that embryogenesis is remarkably robust suggests that additional layers of regulation reinforce patterning. Here, we demonstrate that geometrical confinement orchestrates the spatial organisation of initially randomly positioned subpopulations of spontaneously differentiating mouse embryonic stem cells. We use micropatterning in combination with pharmacological manipulations and quantitative imaging to dissociate the multiple effects of geometry. We show that the positioning of a pre-streak-like population marked by brachyury (T) is decoupled from the size of its population, and that breaking radial symmetry of patterns imposes polarised patterning. We provide evidence for a model in which the overall level of diffusible signals together with the history of the cell culture define the number of T+ cells, whereas geometrical constraints guide patterning in a multi-step process involving a differential response of the cells to multicellular spatial organisation. Our work provides a framework for investigating robustness of patterning and provides insights into how to guide symmetry-breaking events in aggregates of pluripotent cells.


Asunto(s)
Células Madre Embrionarias/citología , Proteínas Fetales/metabolismo , Gastrulación/fisiología , Proteínas de Dominio T Box/metabolismo , Animales , Movimiento Celular/fisiología , Células Cultivadas , Gastrulación/genética , Ratones , Proteína Nodal/metabolismo , Receptores de Factores de Crecimiento de Fibroblastos/metabolismo , Transducción de Señal , Proteínas Wnt/metabolismo
12.
14.
Semin Cell Dev Biol ; 65: 60-68, 2017 05.
Artículo en Inglés | MEDLINE | ID: mdl-27751776

RESUMEN

The proneural gene, Atoh1, is necessary and in some contexts sufficient for early inner ear hair cell development. Its function is the subject of intensive research, not least because of the possibility that it could be used in therapeutic strategies to reverse hair cell loss in deafness. However, it is clear that Atoh1's function is highly context dependent. During inner ear development, Atoh1 is only able to promote hair cell differentiation at specific developmental stages. Outside the ear, Atoh1 is required for differentiation of a variety of other cell types, for example in the intestine and cerebellum. The reasons for this context dependence are poorly understood. So far, the pathways and key players that instruct Atoh1 to act as a mechanosensory cell fate determinant in the context of the inner ear are largely unknown. Here we review evidence that suggests that Atoh1 function in hair cell differentiation is modulated by interaction with other transcription factors. We particularly focus on the possible roles of Gfi1 and Pou4f3, drawing from studies in mouse, Drosophila and C. elegans.


Asunto(s)
Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Proteínas de Unión al ADN/genética , Células Ciliadas Auditivas/metabolismo , Proteínas de Homeodominio/genética , Mecanorreceptores/metabolismo , Factor de Transcripción Brn-3C/genética , Factores de Transcripción/genética , Animales , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Caenorhabditis elegans/citología , Caenorhabditis elegans/crecimiento & desarrollo , Caenorhabditis elegans/metabolismo , Diferenciación Celular , Proteínas de Unión al ADN/metabolismo , Drosophila melanogaster/citología , Drosophila melanogaster/crecimiento & desarrollo , Drosophila melanogaster/metabolismo , Regulación del Desarrollo de la Expresión Génica , Células Ciliadas Auditivas/citología , Proteínas de Homeodominio/metabolismo , Mecanorreceptores/citología , Mecanotransducción Celular , Ratones , Especificidad de Órganos , Factor de Transcripción Brn-3C/metabolismo , Factores de Transcripción/metabolismo
15.
Development ; 141(6): 1209-21, 2014 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-24595287

RESUMEN

During gastrulation, epiblast cells are pluripotent and their fate is thought to be constrained principally by their position. Cell fate is progressively restricted by localised signalling cues from areas including the primitive streak. However, it is unknown whether this restriction accompanies, at the individual cell level, a reduction in potency. Investigation of these early transition events in vitro is possible via the use of epiblast stem cells (EpiSCs), self-renewing pluripotent cell lines equivalent to the postimplantation epiblast. Strikingly, mouse EpiSCs express gastrulation stage regional markers in self-renewing conditions. Here, we examined the differentiation potential of cells expressing such lineage markers. We show that undifferentiated EpiSC cultures contain a major subfraction of cells with reversible early primitive streak characteristics, which is mutually exclusive to a neural-like fraction. Using in vitro differentiation assays and embryo grafting we demonstrate that primitive streak-like EpiSCs are biased towards mesoderm and endoderm fates while retaining pluripotency. The acquisition of primitive streak characteristics by self-renewing EpiSCs is mediated by endogenous Wnt signalling. Elevation of Wnt activity promotes restriction towards primitive streak-associated lineages with mesendodermal and neuromesodermal characteristics. Collectively, our data suggest that EpiSC pluripotency encompasses a range of reversible lineage-biased states reflecting the birth of pioneer lineage precursors from a pool of uncommitted EpiSCs similar to the earliest cell fate restriction events taking place in the gastrula stage epiblast.


Asunto(s)
Estratos Germinativos/citología , Línea Primitiva/citología , Vía de Señalización Wnt , Animales , Diferenciación Celular , Linaje de la Célula , Células Cultivadas , Gástrula/citología , Gástrula/embriología , Gástrula/metabolismo , Gastrulación/fisiología , Estratos Germinativos/embriología , Estratos Germinativos/metabolismo , Ratones , Ratones Transgénicos , Placa Neural/citología , Placa Neural/embriología , Placa Neural/metabolismo , Células Madre Pluripotentes/clasificación , Células Madre Pluripotentes/citología , Células Madre Pluripotentes/metabolismo , Línea Primitiva/embriología
16.
PLoS Biol ; 17(2): e3000180, 2019 02.
Artículo en Inglés | MEDLINE | ID: mdl-30811478
17.
Dev Biol ; 397(1): 56-66, 2015 Jan 01.
Artículo en Inglés | MEDLINE | ID: mdl-25446531

RESUMEN

Gro/TLE proteins (TLE1-4) are a family of transcriptional corepressors acting downstream of multiple signalling pathways. Several TLEs are expressed in a dynamic manner throughout embryonic development and at high levels in embryonic stem cells (ESCs). Here we find that Gro/TLE is not required in ESC for sustaining pluripotency and suppressing differentiation genes, but rather is important for the shutting down of the pluripotency network in differentiation. Consistent with this view, we found that one of the Gro/TLE family, TLE4 is expressed heterogeneously in ESCs in a population that corresponds to a Nanog low subset of ESC culture. TLE4 expression is also increased in response to LIF withdrawal and Fgf/Mek/Erk stimulation. To explore the role of Gro/TLE in more detail we generated an allelic series of knockout ESCs of two TLE genes expressed most dynamically in early differentiation, TLE3 and TLE4. Genetic reduction in TLE dose resulted in an increase in the expression of pluripotency markers and inhibition of ESC differentiation towards both epiblast and endoderm lineages. Overexpression of a drug inducible TLE4 could both rescue TLE3/TLE4 compound phenotypes and induce early expression of endoderm (Hhex-Venus) and neural (Sox1-GFP) reporter genes. Taken together, our results suggest that TLE activity is essential for early differentiation where it acts to suppress the pluripotency network, allowing for the initiation of lineage specific gene expression programs.


Asunto(s)
Proteínas Co-Represoras/fisiología , Células Madre Embrionarias/citología , Regulación del Desarrollo de la Expresión Génica , Células Madre Pluripotentes/citología , Proteínas Represoras/fisiología , Alelos , Animales , Diferenciación Celular , Linaje de la Célula , Proteínas de Unión al ADN/metabolismo , Endodermo/metabolismo , Perfilación de la Expresión Génica , Proteínas Fluorescentes Verdes/metabolismo , Células HEK293 , Humanos , Ratones , Ratones Transgénicos , Modelos Genéticos , Fenotipo , Proteínas Represoras/metabolismo
18.
Development ; 140(20): 4125-8, 2013 Oct.
Artículo en Inglés | MEDLINE | ID: mdl-24086077

RESUMEN

In July 2013, the diverse fields of biology, physics and mathematics converged to discuss 'The Physical Biology of Stem Cells', the subject of the third annual symposium of the Cambridge Stem Cell Institute, UK. Two clear themes resonated throughout the meeting: the new insights gained from advances in the acquisition and interpretation of quantitative data; and the importance of 'thinking outside the nucleus' to consider physical influences on cell fate.


Asunto(s)
Diferenciación Celular , Células Madre/citología , Células Madre/fisiología , Animales , Linaje de la Célula , Morfogénesis , Transducción de Señal
19.
Stem Cells ; 31(8): 1511-22, 2013 Aug.
Artículo en Inglés | MEDLINE | ID: mdl-23649667

RESUMEN

Robust development of the early embryo may benefit from mechanisms that ensure that not all pluripotent cells differentiate at exactly the same time: such mechanisms would build flexibility into the process of lineage allocation. This idea is supported by the observation that pluripotent stem cells differentiate at different rates in vitro. We use a clonal commitment assay to confirm that pluripotent cells commit to differentiate asynchronously even under uniform differentiation conditions. Stochastic variability in expression of the Notch target gene Hes1 has previously been reported to influence neural versus mesodermal differentiation through modulation of Notch activity. Here we report that Hes1 also has an earlier role to delay exit from the pluripotent state into all lineages. The early function of Hes1 to delay differentiation can be explained by an ability of Hes1 to amplify STAT3 responsiveness in a cell-autonomous manner. Variability in Hes1 expression therefore helps to explain why STAT3 responsiveness varies between individual ES cells, and this in turn helps to explain why pluripotent cells commit to differentiate asynchronously.


Asunto(s)
Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Proteínas de Homeodominio/metabolismo , Células Madre Pluripotentes/citología , Células Madre Pluripotentes/metabolismo , Receptores Notch/metabolismo , Factor de Transcripción STAT3/metabolismo , Animales , Diferenciación Celular/fisiología , Regulación hacia Abajo , Humanos , Ratones , Proteína Homeótica Nanog , Transducción de Señal , Factor de Transcripción HES-1 , Transfección
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