Cell Fate Decisions In Development Research Articles

Transcription factor fluctuations underlie cell-to-cell variability in a signaling pathway response.

Stochastic differences among clonal cells can initiate cell fate decisions in development or cause cell-to-cell differences in the responses to drugs or extracellular ligands. One hypothesis is that some of this phenotypic variability is caused by stochastic fluctuations in the activities of transcription factors (TFs). We tested this hypothesis in NIH3T3-CG cells using the response to Hedgehog signaling as a model cellular response. Here, we present evidence for the existence of distinct fast- and slow-responding substates in NIH3T3-CG cells. These two substates have distinct expression profiles, and fluctuations in the Prrx1 TF underlie some of the differences in expression and responsiveness between fast and slow cells. Our results show that fluctuations in TFs can contribute to cell-to-cell differences in Hedgehog signaling.

Hippo pathway and Bonus control developmental cell fate decisions in the Drosophila eye.

The canonical function of the Hippo signaling pathway is the regulation of organ growth. How this pathway controls cell-fate determination is less well understood. Here, we identify a function of the Hippo pathway in cell-fate decisions in the developing Drosophila eye, exerted through the interaction of Yorkie (Yki) with the transcriptional regulator Bonus (Bon), an ortholog of mammalian transcriptional intermediary factor 1/tripartite motif (TIF1/TRIM) family proteins. Instead of controlling tissue growth, Yki and Bon promote epidermal and antennal fates at the expense of the eye fate. Proteomic, transcriptomic, and genetic analyses reveal that Yki and Bon control these cell-fate decisions by recruiting transcriptional and post-transcriptional co-regulators and by repressing Notch target genes and activating epidermal differentiation genes. Our work expands the range of functions and regulatory mechanisms under Hippo pathway control.

Combined modelling of mRNA decay dynamics and single-molecule imaging in the Drosophila embryo uncovers a role for P-bodies in 5' to 3' degradation.

Regulation of mRNA degradation is critical for a diverse array of cellular processes and developmental cell fate decisions. Many methods for determining mRNA half-lives rely on transcriptional inhibition or metabolic labelling. Here, we use a non-invasive method for estimating half-lives for hundreds of mRNAs in the early Drosophila embryo. This approach uses the intronic and exonic reads from a total RNA-seq time series and Gaussian process regression to model the dynamics of premature and mature mRNAs. We show how regulation of mRNA stability is used to establish a range of mature mRNA dynamics during embryogenesis, despite shared transcription profiles. Using single-molecule imaging, we provide evidence that, for the mRNAs tested, there is a correlation between short half-life and mRNA association with P-bodies. Moreover, we detect an enrichment of mRNA 3' ends in P-bodies in the early embryo, consistent with 5' to 3' degradation occurring in P-bodies for at least a subset of mRNAs. We discuss our findings in relation to recently published data suggesting that the primary function of P-bodies in other biological contexts is mRNA storage.

Polycomb Directed Cell Fate Decisions in Development and Cancer.

The polycomb group (PcG) proteins are a subset of transcription regulators highly conserved throughout evolution. Their principal role is to epigenetically modify chromatin landscapes and control the expression of master transcriptional programs to determine cellular identity. The two mayor PcG protein complexes that have been identified in mammals to date are Polycomb Repressive Complex 1 (PRC1) and 2 (PRC2). These protein complexes selectively repress gene expression via the induction of covalent post-translational histone modifications, promoting chromatin structure stabilization. PRC2 catalyzes the histone H3 methylation at lysine 27 (H3K27me1/2/3), inducing heterochromatin structures. This activity is controlled by the formation of a multi-subunit complex, which includes enhancer of zeste (EZH2), embryonic ectoderm development protein (EED), and suppressor of zeste 12 (SUZ12). This review will summarize the latest insights into how PRC2 in mammalian cells regulates transcription to orchestrate the temporal and tissue-specific expression of genes to determine cell identity and cell-fate decisions. We will specifically describe how PRC2 dysregulation in different cell types can promote phenotypic plasticity and/or non-mutational epigenetic reprogramming, inducing the development of highly aggressive epithelial neuroendocrine carcinomas, including prostate, small cell lung, and Merkel cell cancer. With this, EZH2 has emerged as an important actionable therapeutic target in such cancers.

Pioneer factors as master regulators of the epigenome and cell fate.

Pioneer factors are transcription factors with the unique ability to initiate opening of closed chromatin. The stability of cell identity relies on robust mechanisms that maintain the epigenome and chromatin accessibility to transcription factors. Pioneer factors counter these mechanisms to implement new cell fates through binding of DNA target sites in closed chromatin and introduction of active-chromatin histone modifications, primarily at enhancers. As master regulators of enhancer activation, pioneers are thus crucial for the implementation of correct cell fate decisions in development, and as such, they hold tremendous potential for therapy through cellular reprogramming. The power of pioneer factors to reshape the epigenome also presents an Achilles heel, as their misexpression has major pathological consequences, such as in cancer. In this Review, we discuss the emerging mechanisms of pioneer factor functions and their roles in cell fate specification, cellular reprogramming and cancer.

The push and pull of DNA methylation.

The control of DNA methylation guides cell-fate decisions in development and disease

Single-cell genomics to study developmental cell fate decisions in zebrafish.

New developments in single-cell genomics have transformed developmental biology in recent years by enabling systematic analysis of embryonic cell types and differentiation trajectories. Ongoing efforts in experimental and computational method development aim to reveal gene-regulatory mechanisms and to provide additional spatio-temporal information about developmental cell fate decisions. Here, we discuss recent technological developments as well as biological applications of single-cell genomics, with a particular focus on analysis of developmental cell fate decisions. Although the approaches described here are generally applicable to a broad range of model systems, we focus our discussion on applications in zebrafish, which has proven to be a particularly powerful model organism for establishing novel methods in single-cell genomics.

The commitment of barley microspores into embryogenesis correlates with miRNA-directed regulation of members of the SPL, GRF and HD-ZIPIII transcription factor families.

Microspore embryogenesis is a model for developmental plasticity and cell fate decisions. To investigate the role of miRNAs in this development, we sequenced sRNAs and the degradome of barley microspores collected prior to (day 0) and after (days 2 and 5) the application of a stress treatment known to induce embryogenesis. Microspores isolated at these timepoints were uniform in both appearance and in their complements of sRNAs. We detected 68 miRNAs in microspores. The abundance of 51 of these miRNAs differed significantly during microspore development. One group of miRNAs was induced when the stress treatment was applied, prior to being repressed when microspores transitioned to embryogenesis. Another group of miRNAs were up-regulated in day-2 microspores and their abundance remained stable or increased in day-5 microspores, a timepoint at which the first clear indications of the transition toward embryogenesis were visible. Collectively, these miRNAs might play a role in the modulation of the stress response, the repression of gametic development, and/or the gain of embryogenic potential. A degradome analysis allowed us to validate the role of miRNAs in regulating 41 specific transcripts. We showed that the transition of microspores toward the embryogenesis pathway involves miRNA-directed regulation of members of the ARF, SPL, GRF, and HD-ZIPIII transcription factor families. We noted that 41.5% of these targets were shared between day-2 and day-5 microspores while 26.8% were unique to day-5 microspores. The former set may act to disrupt transcripts involved in pollen development while the latter set may drive the commitment to embryogenesis.

Genome-Scale CRISPR Screening for Regulators of Cell Fate Transitions.

Knockout CRISPR screening enables the unbiased discovery of genes with afunctional role in almostanycellular or molecular process of interest. The approach couples a genome-scale library of guide RNA (gRNA), the Cas9 endonuclease, and a faithful phenotypic read-out to systematically identify candidate genes via their loss-of-function effect. Here we provide a detailed description of the CRISPR screen protocol and outline how to apply it to decipher the gene networks that underlie developmental cell fate decisions. As a paradigm we use the in vitro model of cell state transition(s) from naive pluripotency to primordial germ cell (PGC) fate, exploiting the Stella-GFP:Esg1-tdTomato (SGET) mouse ESC line. The principles in this protocol can be readily adapted to characterize lineage regulators for other cell fate models and/or for other species.

Quantifying the interplay between genetic and epigenetic regulations in stem cell development

Waddington epigenetic landscape, as a classic metaphor, has been used to explain cellular development and differentiation. However, it remains challenging to quantify the epigenetic landscape. Especially, a key issue arises as what are the underlying mechanisms for the interplay between genetic and epigenetic regulations to govern cell fate decisions in development. Based on a developmental epigenetic model combining histone modifications and gene regulations, we studied state switching mechanisms of histone modifications for stem cell development, and uncovered corresponding epigenetic landscape. The topography of landscape provides a quantitative measure for the relative stability of different attractors or phenotypes. We showed that histone regulations facilitate the occurrence of intermediate states or multistability. From the epigenetic landscape of stem cell differentiation, we identified key cellular states characterized by attractors, including pluripotent stem cell state, differentiated state and intermediate states. We also quantified representative kinetic transition paths for differentiation, reprogramming and transdifferentiation, which agree well with previous experimental observations. Specifically, previous experiments indicate that transdifferentiation can go through a mixed, unspecific intermediate or progenitor-like state. By calculating the kinetic transition paths, our developmental epigenetic models are able to replicate all these three experimental results, and therefore provide theoretical explanations for these experimental observations. We propose that epigenetic regulations play critical roles on the kinetic transitions for differentiation, reprogramming and transdifferentiation, which also provide a source for the heterogeneity of gene expressions observed in developmental process. Our work provides new insights into the roles of epigenetic modifications on controlling gene expression and stem cell differentiation, and facilitates our mechanistic understanding for the cell fate determinations regarding the interplay between genetic and epigenetic regulations.

Visualizing HIPPO Signaling Components in Mouse Early Embryonic Development.

The HIPPO signaling pathway plays an early and essential role in mammalian embryogenesis. The earliest known roles for HIPPO signaling during mouse development include segregating fetal and extraembryonic lineages and establishing the pluripotent progenitors of embryonic stem (ES) cells. In the mouse early embryo, HIPPO signaling responds to multiple cell biological inputs, including cell polarization, cytoskeleton, and cell environment, to influence gene expression and the first cell fate decisions in development. Methods to monitor and manipulate HIPPO signaling in the mouse early embryo are fundamental to discovering mechanisms regulating pluripotency in vivo, but properties of the early embryo, such as small cell number and spherical architecture, pose unique challenges for signaling pathway analysis. Here, we share approaches for visualizing HIPPO signaling in mouse early embryos. In addition, these methods can be applied to visualize HIPPO signaling in other spherical or cystic structures comprised of relatively few cells, such as organoids, or for the examination of other signaling pathways in these contexts.

Genetic interaction between DNA replication and the Notch signaling pathway.

The Notch pathway is a widely conserved cell signaling system found in most metazoans. In Caenorhabditis elegans, GLP-1/Notch signaling is required for developmental cell fate decisions in multiple contexts. A recent report provides a potential link between DNA replication and Notch-dependent proliferation in the C. elegans germline.

Using single-cell genomics to understand developmental processes and cell fate decisions.

High‐throughput ‐omics techniques have revolutionised biology, allowing for thorough and unbiased characterisation of the molecular states of biological systems. However, cellular decision‐making is inherently a unicellular process to which “bulk” ‐omics techniques are poorly suited, as they capture ensemble averages of cell states. Recently developed single‐cell methods bridge this gap, allowing high‐throughput molecular surveys of individual cells. In this review, we cover core concepts of analysis of single‐cell gene expression data and highlight areas of developmental biology where single‐cell techniques have made important contributions. These include understanding of cell‐to‐cell heterogeneity, the tracing of differentiation pathways, quantification of gene expression from specific alleles, and the future directions of cell lineage tracing and spatial gene expression analysis.

Combinatorial ensemble miRNA target prediction of co-regulation networks with non-prediction data.

MicroRNAs (miRNAs) are key regulators of cell-fate decisions in development and disease with a vast array of target interactions that can be investigated using computational approaches. For this study, we developed metaMIR, a combinatorial approach to identify miRNAs that co-regulate identified subsets of genes from a user-supplied list. We based metaMIR predictions on an improved dataset of human miRNA–target interactions, compiled using a machine-learning-based meta-analysis of established algorithms. Simultaneously, the inverse dataset of negative interactions not likely to occur was extracted to increase classifier performance, as measured using an expansive set of experimentally validated interactions from a variety of sources. In a second differential mode, candidate miRNAs are predicted by indicating genes to be targeted and others to be avoided to potentially increase specificity of results. As an example, we investigate the neural crest, a transient structure in vertebrate development where miRNAs play a pivotal role. Patterns of metaMIR-predicted miRNA regulation alone partially recapitulated functional relationships among genes, and separate differential analysis revealed miRNA candidates that would downregulate components implicated in cancer progression while not targeting tumour suppressors. Such an approach could aid in therapeutic application of miRNAs to reduce unintended effects. The utility is available at http://rna.informatik.uni-freiburg.de/metaMIR/.

Plet1 is an epigenetically regulated cell surface protein that provides essential cues to direct trophoblast stem cell differentiation.

Gene loci that are hypermethylated and repressed in embryonic (ESCs) but hypomethylated and expressed in trophoblast (TSCs) stem cells are very rare and may have particularly important roles in early developmental cell fate decisions, as previously shown for Elf5. Here, we assessed another member of this small group of genes, Placenta Expressed Transcript 1 (Plet1), for its function in establishing trophoblast lineage identity and modulating trophoblast differentiation. We find that Plet1 is tightly repressed by DNA methylation in ESCs but expressed on the cell surface of TSCs and trophoblast giant cells. In hypomethylated ESCs that are prone to acquire some trophoblast characteristics, Plet1 is required to confer a trophoblast-specific gene expression pattern, including up-regulation of Elf5. Plet1 displays an unusual biphasic expression profile during TSC differentiation and thus may be pivotal in balancing trophoblast self-renewal and differentiation. Furthermore, overexpression and CRISPR/Cas9-mediated knockout in TSCs showed that high Plet1 levels favour differentiation towards the trophoblast giant cell lineage, whereas lack of Plet1 preferentially induces syncytiotrophoblast formation. Thus, the endogenous dynamics of Plet1 expression establish important patterning cues within the trophoblast compartment by promoting differentiation towards the syncytiotrophoblast or giant cell pathway in Plet1-low and Plet1-high cells, respectively.

UTX in muscle regeneration--the right dose and the right time.

Precise epigenetic modifications in stem cells control developmental programs and cell fate decisions. In particular, the addition or removal of trimethylation of histone 3 lysine 27 (H3K27me3) at lineage-specific genes has been linked to the repression of gene expression, and a precise balance of methyltransferases and demethylases within cells determines H3K27me3 levels. The demethylase UTX is essential for development and tissue homeostasis; however, a role for UTX in stem cell-mediated tissue regeneration is unknown. In this issue of the JCI, Dilworth and colleagues reveal that UTX and its demethylase activity are required in the muscle stem cell lineage for muscle regeneration in response to injury. Specifically, UTX mediates the removal of H3K27me3 in the promoter of the transcription factor myogenin, which regulates myogenic differentiation. The results of this study provide important insight into the contribution of epigenetic regulation in stem cell-mediated regeneration of adult tissues.

On the Origin of Cells

While non-blood cell lineage has been studied for decades by developmental biologists, only recently has it been considered in disease. This is partly due to a lack of suitable reagents in experimental models, but it is also the result of a failure to understand the ability of cells to move or differentiate in pathological environments. This Editorial gives a quick overview of the Special Issue “Cell Fate Decisions in Development and Disease” and underscores the importance of understanding the mechanisms of cell fate determination and lineage.

Regulation of B Cell to Plasma Cell Transition within the Follicular B Cell Response.

Persistent humoral immunity depends on the follicular B cell response and on the generation of somatically mutated high-affinity plasma cells and memory B cells. Upon activation by an antigen, cognately activated follicular B cells and follicular T helper (TFH ) cells initiate germinal centre (GC) reaction during which high-affinity effector cells are generated. The differentiation of activated follicular B cells into plasma cells and memory B cells is guided by complex selection events, both at the cellular and molecular level. The transition of B cell into a plasma cell during the GC response involves alterations in the microenvironment and developmental state of the cell, which are guided by cell-extrinsic signals. The developmental cell fate decisions in response to these signals are coordinated by cell-intrinsic gene regulatory network functioning at epigenetic, transcriptional and post-transcriptional levels.

Assessing the bipotency of in vitro-derived neuromesodermal progenitors.

Retrospective clonal analysis in the mouse has demonstrated that the posterior spinal cord neurectoderm and paraxial mesoderm share a common bipotent progenitor. These neuromesodermal progenitors (NMPs) are the source of new axial structures during embryonic rostrocaudal axis elongation and are marked by the simultaneous co-expression of the transcription factors T(Brachyury) (T(Bra)) and Sox2. NMP-like cells have recently been derived from pluripotent stem cells in vitro following combined stimulation of Wnt and fibroblast growth factor (FGF) signaling. Under these conditions the majority of cultures consist of T(Bra)/Sox2 co-expressing cells after 48-72 hours of differentiation. Although the capacity of these cells to generate posterior neural and paraxial mesoderm derivatives has been demonstrated at the population level, it is unknown whether a single in vitro-derived NMP can give rise to both neural and mesodermal cells. Here we demonstrate that T(Bra) positive cells obtained from mouse epiblast stem cells (EpiSCs) after culture in NMP-inducing conditions can generate both neural and mesodermal clones. This finding suggests that, similar to their embryonic counterparts, in vitro-derived NMPs are truly bipotent and can thus be exploited as a model for studying the molecular basis of developmental cell fate decisions.

Assessing the bipotency of in vitro-derived neuromesodermal progenitors

Retrospective clonal analysis in the mouse has demonstrated that the posterior spinal cord neurectoderm and paraxial mesoderm share a common bipotent progenitor. These neuromesodermal progenitors (NMPs) are the source of new axial structures during embryonic rostrocaudal axis elongation and are marked by the simultaneous co-expression of the transcription factors T(Brachyury) (T(Bra)) and Sox2. NMP-like cells have recently been derived from pluripotent stem cells in vitro following combined stimulation of Wnt and fibroblast growth factor (FGF) signaling. Under these conditions the majority of cultures consist of T(Bra)/Sox2 co-expressing cells after 48-72 hours of differentiation. Although the capacity of these cells to generate posterior neural and paraxial mesoderm derivatives has been demonstrated at the population level, it is unknown whether a single in vitro-derived NMP can give rise to both neural and mesodermal cells. Here we demonstrate that T(Bra) positive cells obtained from mouse epiblast stem cells (EpiSCs) after culture in NMP-inducing conditions can generate both neural and mesodermal clones. This finding suggests that, similar to their embryonic counterparts, in vitro-derived NMPs are truly bipotent and can thus be exploited as a model for studying the molecular basis of developmental cell fate decisions.