A challenge in biology is to associate molecular differences among progenitor cells with their capacity to generate mature cell types. Here, we used expressed DNA barcodes to clonally trace ...transcriptomes over time and applied this to study fate determination in hematopoiesis. We identified states of primed fate potential and located them on a continuous transcriptional landscape. We identified two routes of monocyte differentiation that leave an imprint on mature cells. Analysis of sister cells also revealed cells to have intrinsic fate biases not detectable by single-cell RNA sequencing. Finally, we benchmarked computational methods of dynamic inference from single-cell snapshots, showing that fate choice occurs earlier than is detected by state-of the-art algorithms and that cells progress steadily through pseudotime with precise and consistent dynamics.
The Hippo-signaling pathway is an important regulator of cellular proliferation and organ size. However, little is known about the role of this cascade in the control of cell fate. Employing a ...combination of lineage tracing, clonal analysis, and organoid culture approaches, we demonstrate that Hippo pathway activity is essential for the maintenance of the differentiated hepatocyte state. Remarkably, acute inactivation of Hippo pathway signaling in vivo is sufficient to dedifferentiate, at very high efficiencies, adult hepatocytes into cells bearing progenitor characteristics. These hepatocyte-derived progenitor cells demonstrate self-renewal and engraftment capacity at the single-cell level. We also identify the NOTCH-signaling pathway as a functional important effector downstream of the Hippo transducer YAP. Our findings uncover a potent role for Hippo/YAP signaling in controlling liver cell fate and reveal an unprecedented level of phenotypic plasticity in mature hepatocytes, which has implications for the understanding and manipulation of liver regeneration.
The Hippo signaling pathway, also known as the Salvador−Warts−Hippo pathway, is a regulator of organ size. The pathway takes its name from the Drosophila protein kinase, Hippo (STK4/MST1 and ...STK3/MST2 in mammals), which, when inactivated, leads to considerable tissue overgrowth. In mammals, MST1 and MST2 negatively regulate the transcriptional co-activators yes-associated protein 1 and WW domain containing transcription regulator 1 (WWTR1/TAZ), which together regulate expression of genes that control proliferation, survival, and differentiation. Yes-associated protein 1 and TAZ activation have been associated with liver development, regeneration, and tumorigenesis. How their activity is dynamically regulated in these contexts is just beginning to be elucidated. We review the mechanisms of Hippo signaling in the liver and explore outstanding questions for future research.
During development and regeneration, proliferation of tissue-specific stem cells is tightly controlled to produce organs of a predetermined size. The molecular determinants of this process remain ...poorly understood. Here, we investigate the function of Yap1, the transcriptional effector of the Hippo signaling pathway, in skin biology. Using gain- and loss-of-function studies, we show that Yap1 is a critical modulator of epidermal stem cell proliferation and tissue expansion. Yap1 mediates this effect through interaction with TEAD transcription factors. Additionally, our studies reveal that α-catenin, a molecule previously implicated in tumor suppression and cell density sensing in the skin, is an upstream negative regulator of Yap1. α-catenin controls Yap1 activity and phosphorylation by modulating its interaction with 14-3-3 and the PP2A phosphatase. Together, these data identify Yap1 as a determinant of the proliferative capacity of epidermal stem cells and as an important effector of a “crowd control” molecular circuitry in mammalian skin.
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► Yap is a critical modulator of epidermal stem cell proliferation ► In epidermis, Yap's function is mediated by TEAD transcription factors ► α-catenin is a cell density sensor regulating Yap activity in the epidermis ► A 14-3-3/α-catenin complex limits PP2A-mediated Yap dephosphorylation
Global downregulation of microRNAs (miRNAs) is commonly observed in human cancers and can have a causative role in tumorigenesis. The mechanisms responsible for this phenomenon remain poorly ...understood. Here, we show that YAP, the downstream target of the tumor-suppressive Hippo-signaling pathway regulates miRNA biogenesis in a cell-density-dependent manner. At low cell density, nuclear YAP binds and sequesters p72 (DDX17), a regulatory component of the miRNA-processing machinery. At high cell density, Hippo-mediated cytoplasmic retention of YAP facilitates p72 association with Microprocessor and binding to a specific sequence motif in pri-miRNAs. Inactivation of the Hippo pathway or expression of constitutively active YAP causes widespread miRNA suppression in cells and tumors and a corresponding posttranscriptional induction of MYC expression. Thus, the Hippo pathway links contact-inhibition regulation to miRNA biogenesis and may be responsible for the widespread miRNA repression observed in cancer.
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•Hippo pathway regulates microRNA biogenesis•YAP sequesters p72 (DDX17) from the Microprocessor in a cell-density-dependent manner•p72 (DDX17) recognizes a sequence motif in pri-miRNAs•YAP mediates widespread microRNA suppression in tumors
The Hippo-signaling pathway and its downstream target YAP regulate miRNA biogenesis in a cell-density-dependent manner, which may be responsible for the widespread miRNA repression commonly observed in human cancers.
Tracing the lineage history of cells is key to answering diverse and fundamental questions in biology. Coupling of cell ancestry information with other molecular readouts represents an important goal ...in the field. Here, we describe the CRISPR array repair lineage tracing (CARLIN) mouse line and corresponding analysis tools that can be used to simultaneously interrogate the lineage and transcriptomic information of single cells in vivo. This model exploits CRISPR technology to generate up to 44,000 transcribed barcodes in an inducible fashion at any point during development or adulthood, is compatible with sequential barcoding, and is fully genetically defined. We have used CARLIN to identify intrinsic biases in the activity of fetal liver hematopoietic stem cell (HSC) clones and to uncover a previously unappreciated clonal bottleneck in the response of HSCs to injury. CARLIN also allows the unbiased identification of transcriptional signatures associated with HSC activity without cell sorting.
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•CARLIN is a stable, genetically defined mouse line for CRISPR-based lineage tracing•Can be activated at any point to generate 44,000 transcribed barcodes across tissues•Sequential, pulsed induction can be used to determine cellular phylogeny in vivo•Heterogeneity in HSC proliferation following myeloablation revealed
CARLIN is a mouse model that allows for simultaneous analysis of lineage and transcriptomic information of single cells in vivo.
Stem cell (SC) activity fluctuates throughout an organism's lifetime to maintain homeostatic conditions in all tissues. As animals develop and age, their organs must remodel and regenerate themselves ...in response to environmental and physiological demands. Recently, the highly conserved Hippo signaling pathway, discovered in Drosophila melanogaster , has been implicated as a key regulator of organ size control across species. Deregulation is associated with substantial overgrowth phenotypes and eventual onset of cancer in various tissues. Importantly, emerging evidence suggests that the Hippo pathway can modulate its effects on tissue size by the direct regulation of SC proliferation and maintenance. These findings provide an attractive model for how this pathway might communicate physiological needs for growth to tissue-specific SC pools. In this review, we summarize the current and emerging data linking Hippo signaling to SC function.
Tissue regeneration is vital to the form and function of an organ. At the core of an organs’ ability to self-renew is the stem cell, which maintains homeostasis, and repopulates injured or aged ...tissue. Tissue damage can dramatically change the dimensions of an organ, and during regeneration, an organ must halt growth once the original tissue dimensions have been restored. Therefore, stem cells must give rise to the appropriate number of differentiated progeny to achieve homeostasis. How this tissue-size checkpoint is regulated and how tissue size information relayed to stem cell compartments is unclear, however, it is likely that these mechanisms are altered during the course of tumorigenesis. An emerging signaling cascade, the Hippo Signaling Pathway, is a broadly conserved potent organ size regulator 1 . However, this pathway does not act alone. A number of examples demonstrate crosstalk between Hippo and other signaling pathways including Wnt, Tgfβ and Notch, with implications for stem cell biology. Here, we focus on these interactions primarily in the context of well characterized stem cell populations.
Summary The Hippo pathway and its regulatory target, YAP, has recently emerged as an important biochemical signaling pathway that tightly governs epithelial tissue growth. Initially defined in ...Drosophilia, this pathway has shown remarkable conservation in vertebrate systems with many components of the Hippo/YAP pathway showing biochemical and functional conservation. The liver is particularly sensitive to changes in Hippo/YAP signaling with rapid increases in liver size becoming manifest on the order of days to weeks after perturbation. The first identified direct targets of Hippo/YAP signaling were pro-proliferative and anti-apoptotic gene programs, but recent work has now implicated this pathway in cell fate choice, stem cell maintenance/renewal, epithelial to mesenchymal transition, and oncogenesis. The mechanisms by which Hippo/YAP signaling is changed endogenously are beginning to come to light as well as how this pathway interacts with other signaling pathways, and important details for designing new therapeutic interventions. This review focuses on the known roles for Hippo/YAP signaling in the liver and promising avenues for future study.
The Hippo/YAP signaling pathway is a crucial regulator of tissue growth, stem cell activity, and tumorigenesis. However, the mechanism by which YAP controls transcription remains to be fully ...elucidated. Here, we utilize global chromatin occupancy analyses to demonstrate that robust YAP binding is restricted to a relatively small number of distal regulatory elements in the genome. YAP occupancy defines a subset of enhancers and superenhancers with the highest transcriptional outputs. YAP modulates transcription from these elements predominantly by regulating promoter-proximal polymerase II (Pol II) pause release. Mechanistically, YAP interacts and recruits the Mediator complex to enhancers, allowing the recruitment of the CDK9 elongating kinase. Genetic and chemical perturbation experiments demonstrate the requirement for Mediator and CDK9 in YAP-driven phenotypes of overgrowth and tumorigenesis. Our results here uncover the molecular mechanisms employed by YAP to exert its growth and oncogenic functions, and suggest strategies for intervention.
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•YAP/TAZ binding is restricted to a subset of distal regulatory regions in the genome•YAP/TAZ occupancy confers potent transcriptional activity to superenhancer regions•YAP/TAZ regulate transcriptional elongation•YAP recruits the Mediator complex and CDK9-elongating kinase
The transcriptional coactivators YAP and TAZ are critical regulators of stem cell activity and tumorigenesis. Galli et al. show that YAP/TAZ binding is restricted to a relatively small number of the most potent enhancers in the genome. They show that YAP/TAZ regulate transcriptional elongation from these elements by recruiting the Mediator complex.
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