RESUMO
The development of intestinal organoids from single adult intestinal stem cells in vitro recapitulates the regenerative capacity of the intestinal epithelium1,2. Here we unravel the mechanisms that orchestrate both organoid formation and the regeneration of intestinal tissue, using an image-based screen to assay an annotated library of compounds. We generate multivariate feature profiles for hundreds of thousands of organoids to quantitatively describe their phenotypic landscape. We then use these phenotypic fingerprints to infer regulatory genetic interactions, establishing a new approach to the mapping of genetic interactions in an emergent system. This allows us to identify genes that regulate cell-fate transitions and maintain the balance between regeneration and homeostasis, unravelling previously unknown roles for several pathways, among them retinoic acid signalling. We then characterize a crucial role for retinoic acid nuclear receptors in controlling exit from the regenerative state and driving enterocyte differentiation. By combining quantitative imaging with RNA sequencing, we show the role of endogenous retinoic acid metabolism in initiating transcriptional programs that guide the cell-fate transitions of intestinal epithelium, and we identify an inhibitor of the retinoid X receptor that improves intestinal regeneration in vivo.
Assuntos
Organoides/citologia , Organoides/fisiologia , Fenótipo , Regeneração/fisiologia , Animais , Diferenciação Celular/efeitos dos fármacos , Diferenciação Celular/genética , Enterócitos/citologia , Enterócitos/efeitos dos fármacos , Homeostase/efeitos dos fármacos , Mucosa Intestinal/efeitos dos fármacos , Mucosa Intestinal/metabolismo , Intestinos/citologia , Intestinos/efeitos dos fármacos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Organoides/efeitos dos fármacos , Organoides/metabolismo , Receptores do Ácido Retinoico/antagonistas & inibidores , Receptores do Ácido Retinoico/metabolismo , Regeneração/efeitos dos fármacos , Análise de Sequência de RNA , Transdução de Sinais/efeitos dos fármacos , Transcrição Gênica/efeitos dos fármacos , Tretinoína/metabolismo , Vitamina A/farmacologiaRESUMO
Mouse embryonic stem cells (mESCs) are biased toward producing embryonic rather than extraembryonic endoderm fates. Here, we identify the mechanism of this barrier and report that the histone deacetylase Hdac3 and the transcriptional corepressor Dax1 cooperatively limit the lineage repertoire of mESCs by silencing an enhancer of the extraembryonic endoderm-specifying transcription factor Gata6. This restriction is opposed by the pluripotency transcription factors Nr5a2 and Esrrb, which promote cell type conversion. Perturbation of the barrier extends mESC potency and allows formation of 3D spheroids that mimic the spatial segregation of embryonic epiblast and extraembryonic endoderm in early embryos. Overall, this study shows that transcriptional repressors stabilize pluripotency by biasing the equilibrium between embryonic and extraembryonic lineages that is hardwired into the mESC transcriptional network.
Assuntos
Receptor Nuclear Órfão DAX-1 , Histona Desacetilases , Células-Tronco Embrionárias Murinas/citologia , Animais , Diferenciação Celular , Células Cultivadas , Receptor Nuclear Órfão DAX-1/genética , Receptor Nuclear Órfão DAX-1/metabolismo , Feminino , Fator de Transcrição GATA6/genética , Histona Desacetilases/genética , Histona Desacetilases/metabolismo , Masculino , Camundongos , RNA Interferente Pequeno/genética , Receptores Citoplasmáticos e Nucleares/genética , Receptores Citoplasmáticos e Nucleares/metabolismo , Receptores de Estrogênio/genética , Receptores de Estrogênio/metabolismoRESUMO
Intestinal organoids are complex three-dimensional structures that mimic the cell-type composition and tissue organization of the intestine by recapitulating the self-organizing ability of cell populations derived from a single intestinal stem cell. Crucial in this process is a first symmetry-breaking event, in which only a fraction of identical cells in a symmetrical sphere differentiate into Paneth cells, which generate the stem-cell niche and lead to asymmetric structures such as the crypts and villi. Here we combine single-cell quantitative genomic and imaging approaches to characterize the development of intestinal organoids from single cells. We show that their development follows a regeneration process that is driven by transient activation of the transcriptional regulator YAP1. Cell-to-cell variability in YAP1, emerging in symmetrical spheres, initiates Notch and DLL1 activation, and drives the symmetry-breaking event and formation of the first Paneth cell. Our findings reveal how single cells exposed to a uniform growth-promoting environment have the intrinsic ability to generate emergent, self-organized behaviour that results in the formation of complex multicellular asymmetric structures.
Assuntos
Intestinos/citologia , Organoides/citologia , Organoides/crescimento & desenvolvimento , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Proteínas de Ligação ao Cálcio , Proteínas de Ciclo Celular , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Camundongos , Organoides/metabolismo , Celulas de Paneth/citologia , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Análise de Célula Única , Proteínas de Sinalização YAPRESUMO
Developmental cell fate specification is a unidirectional process that can be reverted in response to injury or experimental reprogramming. Whether differentiation and de-differentiation trajectories intersect mechanistically is unclear. Here, we performed comparative screening in lineage-related mouse naïve embryonic stem cells (ESCs) and primed epiblast stem cells (EpiSCs), and identified the constitutively expressed zinc finger transcription factor (TF) Zfp281 as a bidirectional regulator of cell state interconversion. We showed that subtle chromatin binding changes in differentiated cells translate into activation of the histone H3 lysine 9 (H3K9) methyltransferase Ehmt1 and stabilization of the zinc finger TF Zic2 at enhancers and promoters. Genetic gain-of-function and loss-of-function experiments confirmed a critical role of Ehmt1 and Zic2 downstream of Zfp281 both in driving exit from the ESC state and in restricting reprogramming of EpiSCs. Our study reveals that cell type-invariant chromatin association of Zfp281 provides an interaction platform for remodeling the cis-regulatory network underlying cellular plasticity.
Assuntos
Diferenciação Celular , Regulação da Expressão Gênica , Histona-Lisina N-Metiltransferase/metabolismo , Células-Tronco Embrionárias Murinas/citologia , Células-Tronco Pluripotentes/citologia , Fatores de Transcrição/metabolismo , Animais , Células Cultivadas , Cromatina/química , Cromatina/metabolismo , Camundongos , Células-Tronco Embrionárias Murinas/metabolismo , Células-Tronco Pluripotentes/metabolismoRESUMO
GDF8, or myostatin, is a member of the TGF-ß superfamily of secreted polypeptide growth factors. GDF8 is a potent negative regulator of myogenesis both in vivo and in vitro. We found that GDF8 signaling was inhibited by the small molecule ATP competitive inhibitors dorsomorphin and LDN-193189. These compounds were previously shown to be potent inhibitors of BMP signaling by binding to the BMP type I receptors ALK1/2/3/6. We present the crystal structure of the type II receptor ActRIIA with dorsomorphin and demonstrate that dorsomorphin or LDN-193189 target GDF8 induced Smad2/3 signaling and repression of myogenic transcription factors. As a result, both inhibitors rescued myogenesis in myoblasts treated with GDF8. As revealed by quantitative live cell microscopy, treatment with dorsomorphin or LDN-193189 promoted the contractile activity of myotubular networks in vitro. We therefore suggest these inhibitors as suitable tools to promote functional myogenesis.
Assuntos
Receptores de Activinas Tipo II/metabolismo , Diferenciação Celular , Mioblastos/efeitos dos fármacos , Miostatina/farmacologia , Pirazóis/farmacologia , Pirimidinas/farmacologia , Transdução de Sinais , Receptores de Activinas Tipo II/química , Sequência de Aminoácidos , Animais , Sítios de Ligação , Humanos , Camundongos , Dados de Sequência Molecular , Mioblastos/citologia , Mioblastos/metabolismo , Ligação Proteica , Células Sf9 , Proteína Smad2/metabolismo , Proteína Smad3/metabolismo , Spodoptera , Fatores de Transcrição/metabolismoRESUMO
BACKGROUND: BMP-induced chemotaxis of mesenchymal progenitors is fundamental for vertebrate development, disease and tissue repair. BMP2 induces Smad and non-Smad signalling. Whereas signal transduction via Smads lead to transcriptional responses, non-Smad signalling induces both, transcriptional and immediate/early non-transcriptional responses. However, the molecular mechanisms by which BMP2 facilitates planar cell polarity, cortical actin rearrangements, lamellipodia formation and chemotaxis of mesenchymal progenitors are poorly understood. Our aim was to uncover the molecular mechanism by which BMP2 facilitates chemotaxis via the BMP2-dependent activation of PI3K and spatiotemporal control of PIP3 production important for actin rearrangements at the mesenchymal cell cytocortex. RESULTS: We unveiled the molecular mechanism by which BMP2 induces non-Smad signalling by PI3K and the role of the second messenger PIP3 in BMP2-induced planar cell polarity, cortical actin reorganisation and lamellipodia formation. By using protein interaction studies, we identified the class Ia PI3K regulatory subunit p55γ to act as a specific and non-redundant binding partner for BMP receptor type II (BMPRII) in concert with the catalytic subunit p110α. We mapped the PI3K interaction to a region within the BMPRII kinase. Either BMP2 stimulation or increasing amounts of BMPRI facilitated p55γ association with BMPRII, but BMPRII kinase activity was not required for the interaction. We visualised BMP2-dependent PIP3 production via PI3K p55γ/p110α and were able to localise PIP3 to the leading edge of intact cells during the process of BMP2-induced planar cell polarity and actin dependent lamellipodia formation. Using mass spectrometry, we found the highly PIP3-sensitive PH-domain protein LL5ß to act as a novel BMP2 effector in orchestrating cortical actin rearrangements. By use of live cell imaging we found that knock-down of p55γ or LL5ß or pharmacological inhibition of PI3K impaired BMP2-induced migratory responses. CONCLUSIONS: Our results provide evidence for an important contribution of the BMP2-PI3K (p55γ/p110α)- PIP3-LL5ß signalling axis in mesenchymal progenitor cell chemotaxis. We demonstrate molecular insights into BMP2-induced PI3K signalling on the level of actin reorganisation at the leading edge cytocortex. These findings are important to better understand BMP2-induced cytoskeletal reorganisation and chemotaxis of mesenchymal progenitors in different physiological or pathophysiological contexts.
Assuntos
Proteína Morfogenética Óssea 2/farmacologia , Proteínas de Transporte/metabolismo , Quimiotaxia/efeitos dos fármacos , Classe Ia de Fosfatidilinositol 3-Quinase/metabolismo , Fosfatos de Inositol/biossíntese , Proteínas de Membrana/metabolismo , Proteínas dos Microfilamentos/metabolismo , Mioblastos/citologia , Mioblastos/enzimologia , Actinas/metabolismo , Sequência de Aminoácidos , Androstadienos/farmacologia , Animais , Receptores de Proteínas Morfogenéticas Ósseas Tipo I , Receptores de Proteínas Morfogenéticas Ósseas Tipo II/metabolismo , Linhagem Celular , Classe Ia de Fosfatidilinositol 3-Quinase/química , Células HEK293 , Humanos , Mesoderma/citologia , Camundongos , Modelos Biológicos , Dados de Sequência Molecular , Mioblastos/efeitos dos fármacos , Mioblastos/metabolismo , Peptídeos/química , Fosforilação/efeitos dos fármacos , Fosfotirosina/metabolismo , Ligação Proteica/efeitos dos fármacos , Pseudópodes/efeitos dos fármacos , Pseudópodes/metabolismo , Transdução de Sinais/efeitos dos fármacos , WortmaninaRESUMO
Gastruloids are 3D structures generated from pluripotent stem cells recapitulating fundamental principles of embryonic pattern formation. Using single-cell genomic analysis, we provide a resource mapping cell states and types during gastruloid development and compare them with the in vivo embryo. We developed a high-throughput handling and imaging pipeline to spatially monitor symmetry breaking during gastruloid development and report an early spatial variability in pluripotency determining a binary response to Wnt activation. Although cells in the gastruloid-core revert to pluripotency, peripheral cells become primitive streak-like. These two populations subsequently break radial symmetry and initiate axial elongation. By performing a compound screen, perturbing thousands of gastruloids, we derive a phenotypic landscape and infer networks of genetic interactions. Finally, using a dual Wnt modulation, we improve the formation of anterior structures in the existing gastruloid model. This work provides a resource to understand how gastruloids develop and generate complex patterns in vitro.
Assuntos
Embrião de Mamíferos , Células-Tronco Pluripotentes , Camundongos , Animais , Embrião de Mamíferos/metabolismo , Linha Primitiva/metabolismo , Desenvolvimento EmbrionárioRESUMO
To promote infections, pathogens exploit host cell machineries such as structural elements of the plasma membrane. Studying these interactions and identifying molecular players are ideal for gaining insights into the fundamental biology of the host cell. Here, we used the anthrax toxin to screen a library of 1,500 regulatory, cell-surface, and membrane trafficking genes for their involvement in the intoxication process. We found that endoplasmic reticulum (ER)-Golgi-localized proteins TMED2 and TMED10 are required for toxin oligomerization at the plasma membrane of human cells, an essential step dependent on localization to cholesterol-rich lipid nanodomains. Biochemical, morphological, and mechanistic analyses showed that TMED2 and TMED10 are essential components of a supercomplex that operates the exchange of both cholesterol and ceramides at ER-Golgi membrane contact sites. Overall, this study of anthrax intoxication led to the discovery that lipid compositional remodeling at ER-Golgi interfaces fully controls the formation of functional membrane nanodomains at the cell surface.
Assuntos
Retículo Endoplasmático , Proteínas de Transporte Nucleocitoplasmático/metabolismo , Proteínas de Transporte Vesicular , Membrana Celular/metabolismo , Ceramidas/metabolismo , Colesterol/metabolismo , Retículo Endoplasmático/metabolismo , Complexo de Golgi/metabolismo , Humanos , Proteínas de Membrana/metabolismo , Proteínas de Transporte Vesicular/metabolismoRESUMO
Organoids recapitulate complex 3D organ structures and represent a unique opportunity to probe the principles of self-organization. While we can alter an organoid's morphology by manipulating the culture conditions, the morphology of an organoid often resembles that of its original organ, suggesting that organoid morphologies are governed by a set of tissue-specific constraints. Here, we establish a framework to identify constraints on an organoid's morphological features by quantifying them from microscopy images of organoids exposed to a range of perturbations. We apply this framework to Madin-Darby canine kidney cysts and show that they obey a number of constraints taking the form of scaling relationships or caps on certain parameters. For example, we found that the number, but not size, of cells increases with increasing cyst size. We also find that these constraints vary with cyst age and can be altered by varying the culture conditions. We observed similar sets of constraints in intestinal organoids. This quantitative framework for identifying constraints on organoid morphologies may inform future efforts to engineer organoids.
Assuntos
Cistos , Organoides , Animais , Cães , FenótipoRESUMO
Image-based phenotypic screening relies on the extraction of multivariate information from cells cultured under a large variety of conditions. Technical advances in high-throughput microscopy enable screening in increasingly complex and biologically relevant model systems. To this end, organoids hold great potential for high-content screening because they recapitulate many aspects of parent tissues and can be derived from patient material. However, screening is substantially more difficult in organoids than in classical cell lines from both technical and analytical standpoints. In this review, we present an overview of studies employing organoids for screening applications. We discuss the promises and challenges of small-molecule treatments in organoids and give practical advice on designing, running, and analyzing high-content organoid-based phenotypic screens.
Assuntos
Organoides , Humanos , Organoides/metabolismoRESUMO
Cells act as building blocks of multicellular organisms, forming higher-order structures at different biological scales. Niches, tissues and, ultimately, entire organisms consist of single cells that remain in constant communication. Emergence of developmental patterns and tissue architecture thus relies on single cells acting as a collective, coordinating growth, migration, cell fate transitions and cell type sorting. For this, information has to be transmitted forward from cells to tissues and fed back to the individual cell to allow dynamic and robust coordination. Here, we define the design principles of tissue organisation integrating chemical, genetic and mechanical cues. We also review the state-of-the-art technologies used for dissecting collective cellular behaviours at single cell- and tissue-level resolution. We finally outline future challenges that lie in a comprehensive understanding of how single cells coordinate across biological scales to insure robust development, homoeostasis and regeneration of tissues.
Assuntos
Especificidade de Órgãos , Animais , Fenômenos Biomecânicos , Fenômenos Eletrofisiológicos , Matriz Extracelular/metabolismo , Homeostase , Humanos , Modelos BiológicosRESUMO
Clathrin-mediated endocytosis, an essential process for plasma membrane homeostasis and cell signaling, is characterized by stunning heterogeneity in the size and lifetime of clathrin-coated endocytic pits (CCPs). If and how CCP growth and lifetime are coupled and how this relates to their physiological function are unknown. We combine computational modeling, automated tracking of CCP dynamics, electron microscopy, and functional rescue experiments to demonstrate that CCP growth and lifetime are closely correlated and mechanistically linked by the early-acting endocytic F-BAR protein FCHo2. FCHo2 assembles at the rim of CCPs to control CCP growth and lifetime by coupling the invagination of early endocytic intermediates to clathrin lattice assembly. Our data suggest a mechanism for the nanoscale control of CCP growth and stability that may similarly apply to other metastable structures in cells.