Generation Of Diverse Cell Types Research Articles

Asymmetric T-cell division: insights from cutting-edge experimental techniques and implications for immunotherapy.

Asymmetric cell division is a fundamental process conserved throughout evolution, employed by both prokaryotic and eukaryotic organisms. Its significance lies in its ability to govern cell fate and facilitate the generation of diverse cell types. Therefore, attaining a detailed mechanistic understanding of asymmetric cell division becomes essential for unraveling the complexities of cell fate determination in both healthy and pathological conditions. However, the role of asymmetric division in T-cell biology has only recently been unveiled. Here, we provide an overview of the T-cell asymmetric division field with the particular emphasis on experimental methods and models with the aim to guide the researchers in the selection of appropriate in vitro/in vivo models to study asymmetric division in T cells. We present a comprehensive investigation into the mechanisms governing the asymmetric division in various T-cell subsets underscoring the importance of the asymmetry in fate-determining factor segregation and transcriptional and epigenetic regulation. Furthermore, the intricate interplay of T-cell receptor signaling and the asymmetric division geometry are explored, shedding light on the spatial organization and the impact on cellular fate.

A transcription factor atlas of directed differentiation

Transcription factors (TFs) regulate gene programs, thereby controlling diverse cellular processes and cell states. To comprehensively understand TFs and the programs they control, we created a barcoded library of all annotated human TF splice isoforms (>3,500) and applied it to build a TF Atlas charting expression profiles of human embryonic stem cells (hESCs) overexpressing each TF at single-cell resolution. We mapped TF-induced expression profiles to reference cell types and validated candidate TFs for generation of diverse cell types, spanning all three germ layers and trophoblasts. Targeted screens with subsets of the library allowed us to create a tailored cellular disease model and integrate mRNA expression and chromatin accessibility data to identify downstream regulators. Finally, we characterized the effects of combinatorial TF overexpression by developing and validating a strategy for predicting combinations of TFs that produce target expression profiles matching reference cell types to accelerate cellular engineering efforts.

Transcription factor antagonism regulates heterogeneity in embryonic stem cell states

Gene expression heterogeneity underlies cell states and contributes to developmental robustness. While heterogeneity can arise from stochastic transcriptional processes, the extent to which it is regulated is unclear. Here, we characterize the regulatory program underlying heterogeneity in murine embryonic stem cell (mESC) states. We identify differentially active and transcribed enhancers (DATEs) across states. DATEs regulate differentially expressed genes and are distinguished by co-binding of transcription factors Klf4 and Zfp281. In contrast to other factors that interact in a positive feedback network stabilizing mESC cell-type identity, Klf4 and Zfp281 drive opposing transcriptional and chromatin programs. Abrogation of factor binding to DATEs dampens variation in gene expression, and factor loss alters kinetics of switching between states. These results show antagonism between factors at enhancers results in gene expression heterogeneity and formation of cell states, with implications for the generation of diverse cell types during development.

Analysis of spontaneous emergence of cell polarity with delayed negative feedback.

Cell polarity refers to spatial di erences in the shape and structure of cells, which leads to the generation of diverse cell types playing di erent roles in biological processes. Cell polarization usually involves the localization of some specific signaling molecules to a proper location of the cell membrane. Recent studies proposed that delayed negative feedback may be important for maintaining the robustness of cell polarization and the observed oscillating behavior of signaling cluster. However, the fundamental mechanisms for achieving cell polarization under negative feedback remain controversial. In this paper, we formulate the cell polarization system as a non-local reaction di usion equation with positive and delayed negative feedback loops. Through the Turing stability analysis, we identify the parameter conditions, including the range of the time delay constant, for achieving cell polarization without any inhomogeneous spatial cues. Also, our numerical results support that by controlling the length of the time delay in negative feedback and the magnitude of positive feedback, the oscillating behavior of signaling cluster can be observed in our simulations.

Genetic and activity-dependent mechanisms underlying interneuron diversity.

The proper construction of neural circuits requires the generation of diverse cell types, their distribution to defined regions, and their specific and appropriate wiring. A major objective in neurobiology has been to understand the molecular determinants that link neural birth to terminal specification and functional connectivity, a task that is especially daunting in the case of cortical interneurons. Considerable evidence supports the idea that an interplay of intrinsic and environmental signalling is crucial to the sequential steps of interneuron specification, including migration, selection of a settling position, morphogenesis and synaptogenesis. However, when and how these influences merge to support the appropriate terminal differentiation of different classes of interneurons remains uncertain. In this Review, we discuss a wealth of recent findings that have advanced our understanding of the developmental mechanisms that contribute to the diversification of interneurons and suggest areas of particular promise for further investigation.

Characterization of Cell-Type-Specific DNA Binding Sites of Plant Transcription Factors Using Chromatin Immunoprecipitation.

The generation of diverse cell types in multicellular organisms often requires the activity of cell-type-specific transcription factors. Understanding where these transcription factors bind in controlling specific cellular programs is critical. However, probing these cell-type-specific factors in vivo with standard chromatin immunoprecipitation (ChIP) assays remains a challenge. We have developed an optimized ChIP assay termed Maximized Objects for Better Enrichment (MOBE)-ChIP, which improves ChIP sensitivity and allows the detection of cell-type-specific signals at a genome-wide scale. Here, I describe the procedure for implementing this method for the study of plant transcription factors. Besides being useful for cell-type-specific studies, MOBE-ChIP can also be employed as a general strategy for enhancing ChIP signals.

Hmga2 regulates self-renewal of retinal progenitors.

In vertebrate retina, histogenesis occurs over an extended period. To sustain the temporal generation of diverse cell types, retinal progenitor cells (RPCs) must self-renew. However, self-renewal and regulation of RPCs remain poorly understood. Here, we demonstrate that cell-extrinsic factors coordinate with the epigenetic regulator high-mobility group AT-hook 2 (Hmga2) to regulate self-renewal of late retinal progenitor cells (RPCs). We observed that a small subset of RPCs was capable of clonal propagation and retained multipotentiality of parents in the presence of endothelial cells (ECs), known self-renewal regulators in various stem cell niches. The self-renewing effects, also observed in vivo, involve multiple intercellular signaling pathways, engaging Hmga2. As progenitors exhaust during retinal development, expression of Hmga2 progressively decreases. Analyses of Hmga2-expression perturbation, in vitro and in vivo, revealed that Hmga2 functionally helps to mediate cell-extrinsic influences on late-retinal progenitor self-renewal. Our results provide a framework for integrating the diverse intercellular influences elicited by epigenetic regulators for self-renewal in a dynamic stem cell niche: the developing vertebrate retina.

Klumpfuss distinguishes stem cells from progenitor cells during asymmetric neuroblast division

Asymmetric stem cell division balances maintenance of the stem cell pool and generation of diverse cell types by simultaneously allowing one daughter progeny to maintain a stem cell fate and its sibling to acquire a progenitor cell identity. A progenitor cell possesses restricted developmental potential, and defects in the regulation of progenitor cell potential can directly impinge on the maintenance of homeostasis and contribute to tumor initiation. Despite their importance, the molecular mechanisms underlying the precise regulation of restricted developmental potential in progenitor cells remain largely unknown. We used the type II neural stem cell (neuroblast) lineage in Drosophila larval brain as a genetic model system to investigate how an intermediate neural progenitor (INP) cell acquires restricted developmental potential. We identify the transcription factor Klumpfuss (Klu) as distinguishing a type II neuroblast from an INP in larval brains. klu functions to maintain the identity of type II neuroblasts, and klu mutant larval brains show progressive loss of type II neuroblasts due to premature differentiation. Consistently, Klu protein is detected in type II neuroblasts but is undetectable in immature INPs. Misexpression of klu triggers immature INPs to revert to type II neuroblasts. In larval brains lacking brain tumor function or exhibiting constitutively activated Notch signaling, removal of klu function prevents the reversion of immature INPs. These results led us to propose that multiple mechanisms converge to exert precise control of klu and distinguish a progenitor cell from its sibling stem cell during asymmetric neuroblast division.

A niche for Drosophila neuroblasts?

Stem cells, which can self-renew and give rise to differentiated daughters, are responsible for the generation of diverse cell types during development and the maintenance of tissue/organ homeostasis in adulthood. Thus, the precise regulation of stem-cell self-renewal and proliferative potential is a key aspect of development. The stem-cell niche confers such control by concentrating localized factors including signaling molecules which favor stem-cell self-renew and regulate stem-cell proliferation in line with developmental programs. In contrast, Drosophila neuroblasts (NBs), often referred to as neural stem cells/progenitors, can undergo asymmetric cell division to self-renew and produce differentiated daughters even in isolation (or in culture). Furthermore, these isolated NBs can also progress through an intrinsically regulated temporal series (of transcription factor expression) to generate diverse cell types in vitro. These data argue that NBs may depend only to a limited extent, if at all, on local environment (a niche) for their maintenance. On the other hand, there is increasing evidence which indicate that the interaction between NBs and their surrounding glia is critical for the control of NB proliferative potential and these glia, in conjunction with systemic regulation, perform the niche function to regulate NB behavior. Thus, these observations emphasize the importance of coordinated local microenvironment (niche activity) and systemic environment (global activity) on the regulation of NB behavior in vivo, and suggest NBs may conform to an alternative stem-cell/progenitor maintenance model.

Notch Regulates the Generation of Diverse Cell Types From the Lateral Lineage of Drosophila Antennal Lobe

:Diverse neuronal cell types arise from the lateral neuroblast in the antennal lobe of Drosophila. The authors show that loss of Notch function from the entire lineage during development leads to an absence of local interneurons (LNs) with a concomitant increase in the number of projection neurons (PNs). The presence of the intracellular domain of Notch was observed within the nucleus of newly born neurons within the neuroblast lineage. This leads to the suggestion that Notch acts in a binary fate decision to determine formation of LNs and PNs, resulting in two distinct hemilineages from the single neuroblast. The observation of nuclear Notch in several neuroblast lineages leads us to speculate that this mechanism is widespread during the developing Drosophila brain.

Modifications of RNA polymerase II are pivotal in regulating gene expression states

The regulation of gene expression programmes is essential for the generation of diverse cell types during development and for adaptation to environmental signals. RNA polymerase II (RNAPII) transcribes genetic information and coordinates the recruitment of accessory proteins that are responsible for the establishment of active chromatin states and transcript maturation. RNAPII is post-translationally modified at active genes during transcription initiation, elongation and termination, and thereby recruits specific histone and RNA modifiers. RNAPII complexes are also located at silent genes in promoter-proximal paused configurations that provide dynamic transcriptional regulation downstream from initiation. In embryonic stem cells, silent developmental regulator genes that are repressed by Polycomb are associated with a form of RNAPII that can elongate through coding regions but that lacks the post-translational modifications that are important for coupling RNA synthesis to co-transcriptional maturation. Here, we discuss the mechanisms through which the transcription of silent genes might be dissociated from productive expression, and the sophisticated interplay between the transcriptional machinery, Polycomb repression and RNA processing.

Cytokinesis in development and disease: variations on a common theme.

Cytokinesis is a crucial step in cell proliferation, and remarkably, it is also an important mechanism for developmental regulation in the generation of diverse cell types in eukaryotic organisms. Successful cytokinesis relies on the assembly and activation of an actomyosin-based contractile ring and membrane deposition/fusion in a spatially and temporally precise manner. As such, the molecular pathways governing cytokinesis are highly complex, involving a large number of components forming intricate interactive networks. The complexity of this system, however, may have also provided a rich platform for evolutionary 'tinkering' to achieve specific morphogenetic and developmental outcomes. Furthermore, failed or altered cytokinesis appears to contribute to the development of cancer in unexpected ways.

The Origin of Phenotypic Heterogeneity in a Clonal Cell Population In Vitro

BackgroundThe spontaneous emergence of phenotypic heterogeneity in clonal populations of mammalian cells in vitro is a rule rather than an exception. We consider two simple, mutually non-exclusive models that explain the generation of diverse cell types in a homogeneous population. In the first model, the phenotypic switch is the consequence of extrinsic factors. Initially identical cells may become different because they encounter different local environments that induce adaptive responses. According to the second model, the phenotypic switch is intrinsic to the cells that may occur even in homogeneous environments.Principal FindingsWe have investigated the “extrinsic” and the “intrinsic” mechanisms using computer simulations and experimentation. First, we simulated in silico the emergence of two cell types in a clonal cell population using a multiagent model. Both mechanisms produced stable phenotypic heterogeneity, but the distribution of the cell types was different. The “intrinsic” model predicted an even distribution of the rare phenotype cells, while in the “extrinsic” model these cells formed small clusters. The key predictions of the two models were confronted with the results obtained experimentally using a myogenic cell line.ConclusionsThe observations emphasize the importance of the “ecological” context and suggest that, consistently with the “extrinsic” model, local stochastic interactions between phenotypically identical cells play a key role in the initiation of phenotypic switch. Nevertheless, the “intrinsic” model also shows some other aspects of reality: The phenotypic switch is not triggered exclusively by the local environmental variations, but also depends to some extent on the phenotypic intrinsic robustness of the cells.

Dare to be different: asymmetric cell division in Drosophila, C. elegans and vertebrates.

One widespread mechanism for the generation of diverse cell types is the unequal inheritance of cell fate determinants. Several such determinants have been identified in the fruitfly Drosophila melanogaster and the worm Caenorhabditis elegans and the molecular machinery responsible for their asymmetric segregation is beginning to be unraveled. To divide asymmetrically, cells establish an axis of polarity, orient the mitotic spindle along this axis and localize cell fate determinants to one side of the cell. During cytokinesis, determinants are then segregated into one of the two daughter cells where they direct cell fate. Here, we outline the steps and factors that are involved in this process in Drosophila and C. elegans and discuss their potential conservation in vertebrates.

Dynamic microtubules lead the way for spindle positioning.

Coordination between the asymmetric partitioning of cell-fate determinants and equal partitioning of genetic material is crucial to the generation of diverse cell types in a developing organism, and to the maintenance of genomic integrity. The emerging model is of a highly organized and dynamic cellular landscape, the form of which is defined by polarized signals within the cell. Cytoskeletal elements are necessary to generate this landscape and to provide motive forces for proper spindle positioning. These forces are generated by interactions between microtubules and the cell cortex.

Control of Shh activity and signaling in the neural tube.

The generation of diverse cell types in the neural tube requires inductive signals that are generally derived from tissues adjacent to the neural tube and capable of patterning cell fates at a distance. Shh, a signaling molecule secreted from the notochord and the floor plate, has been shown to induce motor neurons (MN) as well as interneurons, dorsal to the MN, in a concentration-dependent manner. The cellular response to the Shh signal is mediated by receptors, cytoplasmic factors as well as transcription factors, which act both positively and negatively to modulate Shh activity in the patterning of diverse cell types in the ventral neural tube. Additionally, Shh also cooperates with Bmp and Fgf molecules in the control of diverse neuronal cell fates in the brain.

Murine Delta homologue, mDelta1, expressed on feeder cells controls cellular differentiation.

The Delta/Serrate-Notch pathway is involved in intercellular signaling that controls cell fate during the development of invertebrates and vertebrates. Delta is a prototype of Notch ligands and has been studied extensively in Drosophila. In higher vertebrates, four Delta/Serrate homologues and four Notch homologues have been identified. Recent studies showed that the murine Delta homologue, mDelta1, is essential in early embryogenesis. The biological activity of mammalian Delta and its roles in cellular differentiation, however, have remained unclear. In this study, we first surveyed expression of mDelta1 in the adult mouse and found it to be present in a wide range of tissues. For testing biological activity of mDelta1, we expressed a mDelta1 full-length cDNA in L cells using a eukaryotic expression vector. Effects of mDelta1 on cellular differentiation were examined in two independent systems, featuring C2C12 myogenic differentiation and multipotent murine bone marrow cell differentiation. Inhibition of the former was observed with mDelta1 expression on L cells, associated with suppression of myogenin, a myogenic transcription factor. Expression of mDelta1 in conjunction with GM-CSF promoted differentiation of bone marrow cells to myeloid dendritic cells at the expense of other lineages. Although the effects of mDelta1 on two differentiation systems appeared opposing, as inhibition occurring in one and induction in the other, this can be understood by the unifying concept of generation of diverse cell types from equivalent progenitors. Thus, the present study provided evidence that mammalian Delta participates in intercellular signaling, determining the cell fate in a wide variety of tissues.