Fate Choice Research Articles

Live Tissue Imaging Sheds Light on Cell Level Events During Ectodermal Organ Development.

Embryonic development of ectodermal organs involves a very dynamic range of cellular events and, therefore, requires advanced techniques to visualize them. Ectodermal organogenesis proceeds in well-defined sequential stages mediated by tissue interactions. Different ectodermal organs feature shared morphological characteristics, which are regulated by conserved and reiterative signaling pathways. A wealth of genetic information on the expression patterns and interactions of specific signaling pathways has accumulated over the years. However, the conventional developmental biology methods have mainly relied on two-dimensional tissue histological analyses at fixed time points limiting the possibilities to follow the processes in real time on a single cell resolution. This has complicated the interpretation of cause and effect relationships and mechanisms of the successive events. Whole-mount tissue live imaging approaches are now revealing how reshaping of the epithelial sheet for the initial placodal thickening, budding morphogenesis and beyond, involve coordinated four dimensional changes in cell shapes, well-orchestrated cell movements and specific cell proliferation and apoptosis patterns. It is becoming evident that the interpretation of the reiterative morphogenic signals takes place dynamically at the cellular level. Depending on the context, location, and timing they drive different cell fate choices and cellular interactions regulating a pattern of behaviors that ultimately defines organ shapes and sizes. Here we review how new tissue models, advances in 3D and live tissue imaging techniques have brought new understanding on the cell level behaviors that contribute to the highly dynamic stages of morphogenesis in teeth, hair and related ectodermal organs during development, and in dysplasia contexts.

Investigating higher-order interactions in single-cell data with scHOT.

Single-cell genomics has transformed our ability to examine cell fate choice. Examining cells along a computationally ordered “pseudotime” offers the potential to unpick subtle changes in variability and covariation among key genes. We describe a novel approach, scHOT – single cell Higher Order Testing - which provides a flexible and statistically robust framework for identifying changes in higher order interactions among genes. scHOT can be applied for cells along a continuous trajectory or across space and accommodates any higher order measurement including variability or correlation. We demonstrate the utility of scHOT by studying coordinated changes in higher order interactions during embryonic development of the mouse liver. Additionally, scHOT identifies subtle changes in gene-gene correlations across space using spatially-resolved transcriptomics data from the mouse olfactory bulb. scHOT meaningfully adds to first order differential expression testing and provides a framework for interrogating higher order interactions using single cell data.

Lineage reconstruction from clonal correlations

A central task in developmental biology is to learn the sequence of fate decisions that leads to each mature cell type in a tissue or organism. Recently, clonal labeling of cells using DNA barcodes has emerged as a powerful approach for identifying cells that share a common ancestry of fate decisions. Here we explore the idea that stochasticity of cell fate choice during tissue development could be harnessed to read out lineage relationships after a single step of clonal barcoding. By considering a generalized multitype branching process, we determine the conditions under which the final distribution of barcodes over observed cell types encodes their bona fide lineage relationships. We then propose a method for inferring the order of fate decisions. Our theory predicts a set of symmetries of barcode covariance that serves as a consistency check for the validity of the method. We show that broken symmetries may be used to detect multiple paths of differentiation to the same cell types. We provide computational tools for general use. When applied to barcoding data in hematopoiesis, these tools reconstruct the classical hematopoietic hierarchy and detect couplings between monocytes and dendritic cells and between erythrocytes and basophils that suggest multiple pathways of differentiation for these lineages.

The cytoskeletal regulator HEM1 governs B cell development and prevents autoimmunity

The WAVE regulatory complex (WRC) is crucial for assembly of the peripheral branched actin network constituting one of the main drivers of eukaryotic cell migration. Here, we uncover an essential role of the hematopoietic-specific WRC component HEM1 for immune cell development. Germline-encoded HEM1 deficiency underlies an inborn error of immunity with systemic autoimmunity, at cellular level marked by WRC destabilization, reduced filamentous actin, and failure to assemble lamellipodia. Hem1-/- mice display systemic autoimmunity, phenocopying the human disease. In the absence of Hem1, B cells become deprived of extracellular stimuli necessary to maintain the strength of B cell receptor signaling at a level permissive for survival of non-autoreactive B cells. This shifts the balance of B cell fate choices toward autoreactive B cells and thus autoimmunity.

Conserved and Divergent Features of Adult Neurogenesis in Zebrafish.

Adult neurogenesis, i.e., the generation of neurons from neural stem cells (NSCs) in the adult brain, contributes to brain plasticity in all vertebrates. It varies, however, greatly in extent, location and physiological characteristics between species. During the last decade, the teleost zebrafish (D. rerio) was increasingly used to study the molecular and cellular properties of adult NSCs, in particular as a prominent NSC population was discovered at the ventricular surface of the dorsal telencephalon (pallium), in territories homologous to the adult neurogenic niches of rodents. This model, for its specific features (large NSC population, amenability to intravital imaging, high regenerative capacity) allowed rapid progress in the characterization of basic adult NSC features. We review here these findings, with specific comparisons with the situation in rodents. We specifically discuss the cellular nature of NSCs (astroglial or neuroepithelial cells), their heterogeneities and their neurogenic lineages, and the mechanisms controlling NSC quiescence and fate choices, which all impact the neurogenic output. We further discuss the regulation of NSC activity in response to physiological triggers and non-physiological conditions such as regenerative contexts.

Histone Acetyltransferase MOF Blocks Acquisition of Quiescence in Ground-State ESCs through Activating Fatty Acid Oxidation.

Self-renewing embryonic stem cells (ESCs) respond to environmental cues by exiting pluripotency or entering a quiescent state. The molecular basis underlying this fate choice remains unclear. Here, we show that histone acetyltransferase MOF plays a critical role in this process through directly activating fatty acid oxidation (FAO) in the ground-state ESCs. We further show that the ground-state ESCs particularly rely on elevated FAO for oxidative phosphorylation (OXPHOS) and energy production. Mof deletion or FAO inhibition induces bona fide quiescent ground-state ESCs with an intact core pluripotency network and transcriptome signatures akin to the diapaused epiblasts invivo. Mechanistically, MOF/FAO inhibition acts through reducing mitochondrial respiration (i.e., OXPHOS), which in turn triggers reversible pluripotent quiescence specifically in the ground-state ESCs. The inhibition of FAO/OXPHOS also induces quiescence in naive human ESCs. Our study suggests a general function of the MOF/FAO/OXPHOS axis in regulating cell fate determination in stem cells.

Prdm1 overexpression causes a photoreceptor fate-shift in nascent, but not mature, bipolar cells

The transcription factors Prdm1 (Blimp1) and Vsx2 (Chx10) work downstream of Otx2 to regulate photoreceptor and bipolar cell fates in the developing retina. Mice that lack Vsx2 fail to form bipolar cells while Prdm1 mutants form excess bipolars at the direct expense of photoreceptors. Excess bipolars in Prdm1 mutants appear to derive from rods, suggesting that photoreceptor fate remains mutable for some time after cells become specified. Here we tested whether bipolar cell fate is also plastic during development. To do this, we created a system to conditionally misexpress Prdm1 at different stages of bipolar cell development. We found that Prdm1 blocks bipolar cell formation if expressed before the fate choice decision occurred. When we misexpressed Prdm1 just after the decision to become a bipolar cell was made, some cells were reprogrammed into photoreceptors. In contrast, Prdm1 misexpression in mature bipolar cells did not affect cell fate. We also provide evidence that sustained misexpression of Prdm1 was selectively toxic to photoreceptors. Our data show that bipolar fate is malleable, but only for a short temporal window following fate specification. Prdm1 and Vsx2 act by stabilizing photoreceptor and bipolar fates in developing OTX2+ cells of the retina.

Alignment of Cell Lineage Trees Elucidates Genetic Programs for the Development and Evolution of Cell Types

SummaryA full understanding of the developmental process requires fine-scale characterization of cell divisions and cell types, which are naturally organized as the developmental cell lineage tree (CLT). Technological breakthroughs facilitated determination of more CLTs, but complete comprehension of the data remains difficult without quantitative comparison among CLTs. We hereby quantified phenotypic similarity between CLTs using a novel computational method that exhaustively searches for optimal correspondence between individual cells meanwhile retaining their topological relationships. The revealed CLT similarities allowed us to infer functional similarity at the transcriptome level, identify cell fate transformations, predict functional relationships between mutants, and find evolutionary correspondence between cell types of different species. By allowing quantitative comparison between CLTs, our work is expected to greatly enhance the interpretability of relevant data and help answer the myriad of questions surrounding the developmental process.

Modulation of β-catenin levels regulates cranial neural crest patterning and dispersal into first pharyngeal arch.

Vertebrate cranial neural crest cells (CNCCs) are multipotent, proximal to the source CNCC form the cranial ganglia. Distally, in the pharyngeal arches, they give rise to the craniofacial skeleton and connective tissues. Fate choices are made as CNCC pattern into distinct destination compartments. In spite of this importance, the mechanism patterning CNCC is poorly defined. Here, we report that a novel β-catenin-dependent regulation of N-Cadherin levels may drive CNCC patterning. In mouse embryos, at the first pharyngeal arch axial level, membrane β-catenin levels correlate with the extent of N-cadherin-mediated adhesion and thus suggest the presence of collective and dispersed states of CNCC. Using in vitro human neural crest model and chemical modulators of β-catenin levels, we show a requirement for down-modulating β-catenin for regulating N-cadherin levels and cell-cell adhesion. Similarly, in β-catenin gain-of-function mutant mouse embryos, CNCC fail to lower N-cadherin levels. This indicates a failure to reduce cell-cell adhesion, which may underlie the failure of mutant CNCC to populate first pharyngeal arch. We suggest that β-catenin-mediated regulation of CNCC adhesion, a previously underappreciated mechanism, underlies the patterning of CNCC into fate-specific compartments.

Selective Translation of Cell Fate Regulators Mediates Tolerance to Broad Oncogenic Stress.

Human skin tolerates a surprisingly high burden of oncogenic lesions. Although adult epidermis can suppress the expansion of individual mutant clones, the mechanisms behind tolerance to oncogene activation across broader regions of tissue are unclear. Here, we uncover a dynamic translational mechanism that coordinates oncogenic HRAS-induced hyperproliferation with loss of progenitor self-renewal to restrain aberrant growth and tumorigenesis. We identify translation initiator eIF2B5 as a central co-regulator of HRAS proliferation and cell fate choice. By coupling invivo ribosome profiling with genetic screening, we provide direct evidence that oncogene-induced loss of progenitor self-renewal is driven by eIF2B5-mediated translation of ubiquitination genes. Ubiquitin ligase FBXO32 specifically inhibits epidermal renewal without affecting overall proliferation, thus restraining HRAS-driven tumorigenesis while maintaining normal tissue growth. Thus, oncogene-driven translation is not necessarily inherently tumor promoting but instead can manage widespread oncogenic stress by steering progenitor fate to prolong normal tissue growth.

Three-dimensional niche stiffness synergizes with Wnt7a to modulate the extent of satellite cell symmetric self-renewal divisions.

Satellite cells (SCs), the resident adult stem cells of skeletal muscle, are required for tissue repair throughout life. While many signaling pathways are known to control SC self-renewal, less is known about the mechanisms underlying the spatiotemporal control of self-renewal during skeletal muscle repair. Here, we measured biomechanical changes that accompany skeletal muscle regeneration and determined the implications on SC fate. Using atomic force microscopy, we quantified a 2.9-fold stiffening of the SC niche at time-points associated with planar-oriented symmetric self-renewal divisions. Immunohistochemical analysis confirms increased extracellular matrix deposition within the basal lamina. To test whether three-dimensional (3D) niche stiffness can alter SC behavior or fate, we embedded isolated SC-associated muscle fibers within biochemically inert agarose gels tuned to mimic native tissue stiffness. Time-lapse microscopy revealed that a stiff 3D niche significantly increased the proportion of planar-oriented divisions, without effecting SC viability, fibronectin deposition, or fate change. We then found that 3D niche stiffness synergizes with WNT7a, a biomolecule shown to control SC symmetric self-renewal divisions via the noncanonical WNT/planar cell polarity pathway, to modify stem cell pool expansion. Our results provide new insights into the role of 3D niche biomechanics in regulating SC fate choice.

Beneficial Effects of Blueberry Extracts on Adult Human Neural Progenitor Cells

Abstract Objectives Aging-induced declines in neurogenesis and stem cell functions play a key role in the pathophysiology of age-associated neuronal disorders, and oxidative stress and inflammation are the major contributing factors. Blueberries, rich in polyphenols, have been shown to decrease oxidative stress, inflammation, and improve cognitive function in aged animals and humans. The current study investigated the effects of blueberry (BB) treatments on the viability, proliferation, fate choice, and expression of oxidative stress and inflammation markers of adult human neural progenitor cells (AHNPs), to establish mechanisms underlying the beneficial effects. Methods AHNPs derived from control and Parkinson's disease (PD) patients were pre-treated with various concentrations of freeze-dried BB extract for 7–10 days. Their viability and proliferation were examined using Trypan Blue exclusion and ethynyl-deoxyuridine (EdU) assay, respectively. Some treated cells were labeled with neuronal markers to examine their differentiation. Expression of oxidative stress and inflammation markers such as iNOS and Nox-2 was evaluated using western blot analysis. Whether BB could protect AHNPs from stress induced by dopamine (DA) was investigated by exposing cells to DA for 2–4 hours prior to evaluating their viability, proliferation, calcium buffering, and expression of oxidative stress and inflammation markers. Results Our data show that while BB treatment did not significantly affect the viability of control- and PD-AHNPs, BB extract significantly increased the proliferation rate of PD-AHNPs at higher concentrations. Additionally, BB treatment was able to revive the decrease in cell viability and proliferation following cellular stress induced by DA, indicating a potential neuroprotective effect. BB treatment was also able to protect against the harmful effects on Ca2+ buffering and reduce the increased expression levels of both iNOS and Nox-2 induced by DA exposure. Conclusions Blueberry extracts confer anti-oxidative, anti-inflammatory and neuroprotective effects on control and Parkinson's AHNPs, suggesting a role for dietary nutrients in helping to slow progression of neural dysfunction and neurodegenerative disease during aging. Funding Sources Supported by USDA/ARS intramural grant, and the Robert and Margaret Patricelli Family Foundation.

APPL2 Negatively Regulates Olfactory Functions by Switching Fate Commitments of Neural Stem Cells in Adult Olfactory Bulb via Interaction with Notch1 Signaling.

Adult olfactory neurogenesis plays critical roles in maintaining olfactory functions. Newly-generated neurons in the subventricular zone migrate to the olfactory bulb (OB) and determine olfactory discrimination, but the mechanisms underlying the regulation of olfactory neurogenesis remain unclear. Our previous study indicated the potential of APPL2 (adaptor protein, phosphotyrosine interacting with PH domain and leucine zipper 2) as a modulating factor for neurogenesis in the adult olfactory system. In the present study, we report how APPL2 affects neurogenesis in the OB and thereby mediates olfactory discrimination by using both in vitro neural stem cells (NSCs) and an in vivo animal model-APPL2 transgenic (Tg) mice. In the in vitro study, we found that APPL2 overexpression resulted in NSCs switching from neuronal differentiation to gliogenesis while APPL2 knockdown promoted neurogenesis. In the in vivo study, APPL2 Tg mice had a higher population of glial cells and dampened neuronal production in the olfactory system, including the corpus callosum, OB, and rostral migratory stream. Adult APPL2 Tg mice displayed impaired performance in olfactory discrimination tests compared with wild-type mice. Furthermore, we found that an interaction of APPL2 with Notch1 contributed to the roles of APPL2 in modulating the neurogenic lineage-switching and olfactory behaviors. In conclusion, APPL2 controls olfactory discrimination by switching the fate choice of NSCs via interaction with Notch1 signaling.

Assembly of lung progenitors into developmentally-inspired geometry drives differentiation via cellular tension

During organogenesis groups of differentiating cells self-organize into a series of structural intermediates with defined architectural forms. Evidence is emerging that such architectural forms are important in guiding cell fate, yet in vitro methods to guide cell fate have focused primarily on un-patterned exposure of stems cells to developmentally relevant chemical cues. We set out to ask if organizing differentiating lung progenitors into developmentally relevant structures could be used to influence differentiation status. Specifically, we use elastomeric substrates to guide self-assembly of human pluripotent stem cell-derived lung progenitors into developmentally-relevant sized tubes and assess the impact on differentiation. Culture in 100 μm tubes reduced the percentage of SOX2+SOX9+ cells and reduced proximal fate potential compared to culture in 400 μm tubes or on flat surfaces. Cells in 100 μm tubes curved to conform to the tube surface and experienced increased cellular tension and reduced elongation. Pharmacologic disruption of tension through inhibition of ROCK, myosin II activity and actin polymerization in tubes resulted in maintenance of SOX2+SOX9+ populations. Furthermore, this effect required canonical WNT signaling. This data suggests that structural forms, when developmentally relevant, can drive fate choice during directed differentiation via a tension-based canonical WNT dependent mechanism.

STRIPAK-PP2A regulates Hippo-Yorkie signaling to suppress retinal fate in the Drosophila eye disc peripodial epithelium.

The specification of organs, tissues and cell types results from cell fate restrictions enacted by nuclear transcription factors under the control of conserved signaling pathways. The progenitor epithelium of the Drosophila compound eye, the eye imaginal disc, is a premier model for the study of such processes. Early in development, apposing cells of the eye disc are established as either retinal progenitors or support cells of the peripodial epithelium (PE), in a process whose genetic and mechanistic determinants are poorly understood. We have identified protein phosphatase 2A (PP2A), and specifically a STRIPAK-PP2A complex that includes the scaffolding and substrate-specificity components Cka, Strip and SLMAP, as a critical player in the retina-PE fate choice. We show that these factors suppress ectopic retina formation in the presumptive PE and do so via the Hippo signaling axis. STRIPAK-PP2A negatively regulates Hippo kinase, and consequently its substrate Warts, to release the transcriptional co-activator Yorkie into the nucleus. Thus, a modular higher-order PP2A complex refines the activity of this general phosphatase to act in a precise specification of cell fate.

DDX3X Sits at the Crossroads of Liquid–Liquid and Prionoid Phase Transitions Arbitrating Life and Death Cell Fate Decisions in Stressed Cells

The crosstalk between cellular stress responses and innate immune signaling pathways remains poorly understood. Cells can respond to stressors by assembling stress granules that store 40S ribosomes, translation initiation factors, and mRNAs, and allow the cell to survive. Some stressors can activate the NLRP3 inflammasome, which leads to pyroptotic cell death. Stress granules and the NLRP3 inflammasome provide distinct cell fate choices to the cell. These complexes also involve distinct types of phase transitions-liquid-liquid phase separation for stress granules and prionoid phase transition for the NLRP3 inflammasome. We recently reported that DDX3X modulates this crosstalk by acting as a common essential factor for NLRP3 inflammasome activation and stress granule assembly. Here, we discuss the role of DDX3X in modulating the liquid-liquid phase separation and prionoid phase transition required for making cell fate decisions under stress conditions.

NFI transcription factors provide chromatin access to maintain stem cell identity while preventing unintended lineage fate choices.

Tissue homeostasis and regeneration rely upon resident stem cells (SCs), whose behavior is regulated through niche-dependent crosstalk. The mechanisms underlying SC identity are still unfolding. Here, using spatiotemporal gene ablation in murine hair follicles (HFs), we uncover a critical role for transcription factors (TFs) NFIB and NFIX in maintaining SC identity. Without NFI-TFs, SCs lose hair-regenerating capability, and produce skin bearing striking resemblance to irreversible human alopecia, which also displays reduced NFIs. Through single cell transcriptomics, ATAC-seq and ChIP-seq profiling, we expose a key role for NFIB/NFIX in governing super-enhancer maintenance of the key HF-SC specific TF genes. When NFIB/NFIX are genetically removed, the stemness epigenetic landscape is lost. Super-enhancers driving SC identity are decommissioned, while unwanted lineages are de-repressed ectopically. Together, our findings expose NFIB/NFIX as crucial rheostats of tissue homeostasis, functioning to safeguard the SC epigenome from a breach in lineage confinement that otherwise triggers irreversible tissue degeneration.

Differentiation of transplanted haematopoietic stem cells tracked by single-cell transcriptomic analysis.

How transplanted haematopoietic stem cells (HSCs) behave soon after they reside in a preconditioned host has not been studied due to technical limitations. Here, using single-cell RNA sequencing, we first obtained the transcriptome-based classifications of 28 haematopoietic cell types. We then applied them in conjunction with functional assays to track the dynamic changes of immunophenotypically purified HSCs in irradiated recipients within the first week after transplantation. Based on our transcriptional classifications, most homed HSCs in bone marrow and spleen became multipotent progenitors and, occasionally, some HSCs gave rise to megakaryocytic-erythroid or myeloid precursors. Parallel in vitro and in vivo functional experiments supported the paradigm of robust differentiation without substantial HSC expansion during the first week. Therefore, this study uncovers the previously inaccessible kinetics and fate choices of transplanted HSCs in myeloablated recipients at early stage, with implications for clinical applications of HSCs and other stem cells.

WNT/β-catenin directed metabolic choice uncouples lung-resident alveolar macrophage inflammatory activity from self-renewal

Abstract Fetal-derived tissue-resident macrophages exhibit stem cell-like features of self-renewal in adulthood to maintain macrophage population during homeostasis and/or various insults. However, little is known about the cellular and molecular mechanisms modulating proliferative and inflammatory fate decisions of tissue-resident macrophages in vivo. Here, we show that WNT-β-catenin signaling inhibited lung-resident alveolar macrophage (AM) self-renewal, while simultaneously promoted AM inflammatory activities in vitro and in vivo during influenza virus infection. Mechanistically, WNT engagement facilitated the binding of β-catenin with HIF-1α over its conventional binding partner TCF-4. Such a binding choice led to the elevated macrophage inflammation in a glycolysis-dependent manner, while inhibited AM self-renewal by causing mitochondrial damage and impairing oxidative phosphorylation. Thus, AM self-renewal and inflammatory activity are uncoupled by WNT-β-catenin signaling through HIF-1α-mediated cellular metabolic choice. Importantly, we showed that AM with high HIF-1α activity had limited proliferative capacity and produced inflammatory cytokines, while AM with low HIF-1α activity were highly proliferative and expressed genes associated with tissue repair function in vivo during influenza virus infection. In accordance, we demonstrated that AM proliferation and repopulation were needed for optimal lung repair following the clearance of influenza virus in the respiratory tract. Our results have revealed key mechanisms modulating macrophage fate choice between progeny production versus inflammatory effector activity, and subsequent effects on tissue inflammation and repair.

DDX3X Cellular Valency Allows Stressed Cells to Make a Live or Die Cell Fate Decision by Regulating Stress Granules and the NLRP3 Inflammasome

Abstract Cells of the immune system have to respond to a variety of stressors to help maintain organismal homeostasis. Stress sensing can lead to the assembly of a cytoplasmic membraneless compartment called stress granules (SGs) that helps the cells survive. Cellular stress can also lead to activation of the NLRP3 inflammasome and induction of pyroptosis – a pro-inflammatory programmed cell death. Thus SGs and the NLRP3 inflammasome provide contrasting cell fate choices to a stressed cell – cell survival or pyroptosis. DDX3X is an integral component of SGs [1]. We show that DDX3X promotes the NLRP3 inflammasome activation and ASC speck assembly[2]. Our data suggests that induction of SGs inhibits the NLRP3 inflammasome activation by sequestering the DDX3X molecules. It also suggests that SGs and the NLRP3 inflammasome compete for DDX3X molecules, which makes DDX3X cellular valency critical for this decision making process[3]. Using LPS mediated peritonitis model, we further show that DDX3X promotes and SGs inhibit the NLRP3 inflammasome activation in vivo. Our data points towards a mechanistic paradigm where cells can exploit the cellular valency of biomolecules and a competition between biological processes for common essential factors to make cell fate decisions.