Unique Cell Fates Research Articles

In Vitro Analysis of Thymocyte Signaling.

When a developing thymocyte expresses a TCR, it is subjected to numerous interactions with self-peptide/MHC complexes that determine its fate. These include death by neglect, negative selection (apoptosis and lineage deviation), positive selection, and lineage commitment. Identifying signals that govern these unique cell fates requires the ability to assess the activity, level of expression, subcellular location, and molecular associations between numerous proteins within the developing T cell. Given the unique, temporal, and developmental changes that occur during development, isolating and analyzing small populations of thymocytes are necessary to get a complete picture of the development process. Thus, this chapter describes methods designed to analyze thymocyte signaling under various types of peptide-based stimulation in vitro.

Fetal Thymic Organ Culture and Negative Selection.

Negative selection removes potentially harmful T cell precursors from the conventional T cell pool. This process can involve the induction of apoptosis, anergy, receptor editing, or deviation into a regulatory T cell lineage. As such, this process is essential for the health of an organism through its contribution to central and peripheral tolerance. While a great deal is known about the process, the precise mechanisms that regulate these various forms of negative selection are not clear. Numerous models exist with the potential to address these questions in vitro and in vivo. This chapter describes fetal thymic organ culture methods designed to analyze the signals that determine these unique cell fates.

RNA sequencing of single allergen-specific memory B cells after grass pollen immunotherapy: Two unique cell fates and CD29 as a biomarker for treatment effect.

Sublingual immunotherapy (SLIT) for grass pollen allergy can modify the natural history of allergic rhinitis and is associated with increased allergen-specific IgG4 . IgG4 competitively inhibits functional IgE on the surface of effector cells, such as mast cells and basophils, from binding to allergens. To further understand the important role memory B-cell (Bmem) responses play in mediating the beneficial effects of SLIT, we assessed changes in allergen-specific Bmem subsets induced by SLIT for grass pollen allergy. Blood samples were collected twice outside the pollen season from twenty-seven patients with sensitization to ryegrass pollen (RGP; Lolium perenne) and seasonal rhinoconjunctivitis. Thirteen received 4-month pre-seasonal SLIT for grass pollen allergy, and 14 received standard pharmacotherapy only. Single-cell RNA sequencing was performed on FACS-purified Lol p 1-specific Bmem before and after SLIT from four patients, and significant genes were validated by flow cytometry on the total cohort. Four months of SLIT increased RGP-specific IgE and IgG4 in serum and induced two Lol p 1-specific Bmem subsets with unique transcriptional profiles. Both subsets had upregulated expression of beta 1 integrin ITGB1 (CD29), whereas IGHE (IgE), IGHG4 (IgG4 ), FCER2 (CD23), and IL13RA1 were upregulated in one subset. There was an increase in the proportion of Lol p 1+ Bmem expressing surface IgG4 , CD23, and CD29 after SLIT. A clinically successful 4months course of SLIT for grass pollen allergy induces two transcriptionally unique Bmem fates. Associated changes in surface-expressed proteins on these Bmem subsets can be used as early biomarkers for treatment effects.

Next Generation Cell Culture Tools Featuring Micro‐ and Nanotopographies for Biological Screening (Adv. Funct. Mater. 3/2022)

High-Throughput Screening In article number 2100881, Nicolas H. Voelcker, Jessica E. Frith, Victor J. Cadarso, and co-workers demonstrate a novel approach to imprint micro and nanoscaled topographical features into conventional cell cultureware, facilitating its compatibility with standard biological techniques. This enables high-throughput screening to integrate the effects of surface topographies into unique cell specific responses and fate determination.

Duplication of spiralian-specific TALE genes and evolution of the blastomere specification mechanism in the bivalve lineage

BackgroundDespite the conserved pattern of the cell-fate map among spiralians, bivalves display several modified characteristics during their early development, including early specification of the D blastomere by the cytoplasmic content, as well as the distinctive fate of the 2d blastomere. However, it is unclear what changes in gene regulatory mechanisms led to such changes in cell specification patterns. Spiralian-TALE (SPILE) genes are a group of spiralian-specific transcription factors that play a role in specifying blastomere cell fates during early development in limpets. We hypothesised that the expansion of SPILE gene repertoires influenced the evolution of the specification pattern of blastomere cell fates.ResultsWe performed a transcriptome analysis of early development in the purplish bifurcate mussel and identified 13 SPILE genes. Phylogenetic analysis of the SPILE gene in molluscs suggested that duplications of SPILE genes occurred in the bivalve lineage. We examined the expression patterns of the SPILE gene in mussels and found that some SPILE genes were expressed in quartet-specific patterns, as observed in limpets. Furthermore, we found that several SPILE genes that had undergone gene duplication were specifically expressed in the D quadrant, C and D quadrants or the 2d blastomere. These expression patterns were distinct from the expression patterns of SPILE in their limpet counterparts.ConclusionsThese results suggest that, in addition to their ancestral role in quartet specification, certain SPILE genes in mussels contribute to the specification of the C and D quadrants. We suggest that the expansion of SPILE genes in the bivalve lineage contributed to the evolution of a unique cell fate specification pattern in bivalves.

Comparative Evaluation of Hormones and Hormone-Like Molecule in Lineage Specification of Human Induced Pluripotent Stem Cells.

Background and ObjectivesProficient differentiation of human pluripotent stem cells (hPSCs) into specific lineages is required for applications in regenerative medicine. A growing amount of evidences had implicated hormones and hormone-like molecules as critical regulators of proliferation and lineage specification during in vivo development. Therefore, a deeper understanding of the hormones and hormone-like molecules involved in cell fate decisions is critical for efficient and controlled differentiation of hPSCs into specific lineages. Thus, we functionally and quantitatively compared the effects of diverse hormones (estradiol 17-β (E2), progesterone (P4), and dexamethasone (DM)) and a hormone-like molecule (retinoic acid (RA)) on the regulation of hematopoietic and neural lineage specification.Methods and ResultsWe used 10 nM E2, 3 μM P4, 10 nM DM, and 10 nM RA based on their functional in vivo developmental potential. The sex hormone E2 enhanced functional activity of hematopoietic progenitors compared to P4 and DM, whereas RA impaired hematopoietic differentiation. In addition, E2 increased CD34+CD45+ cells with progenitor functions, even in the CD43− population, a well-known hemogenic marker. RA exhibited lineage-biased potential, preferentially committing hPSCs toward the neural lineage while restricting the hematopoietic fate decision.ConclusionsOur findings reveal unique cell fate potentials of E2 and RA treatment and provide valuable differentiation information that is essential for hPSC applications.

Sean Wu.

Sean Wu.

SUN-004 Unique Cell Fates and Metastatic Phenotypes Are Activated by Progesterone Receptor (PR) Isoform-specific Gene Expression in Fallopian Tube Models of HGSC

High grade serous ovarian carcinoma (HGSC), the most prevalent and aggressive form of ovarian cancer, contains abundant receptors for the ovarian steroid hormones, estrogen (ER; 76%) and progesterone (PR; 35%). These receptors are known to contribute to breast and reproductive cancer development. Our understanding, however, of the mechanisms of PR isoform action in the initiation and progression of ovarian cancer is limited. HGSC is thought to originate from the fallopian tube epithelium (FTE), and accumulating evidence suggests that serous tubal intraepithelial carcinomas (STICs) are precursor lesions to most HGSCs. Our IHC analysis of normal FTE and STICs showed expression of both total PR and activated phopho-Ser294 PR (p-PR). Interestingly, STIC lesions had greater intensity of focal nuclear p-PR, indicative of highly active transcriptional complexes. To investigate PR isoform signaling in early stage HGSC, we utilized p53-dominant negative mutant FTE cells to generate stable cell lines expressing either the PR-A or PR-B isoform. Progestin (progesterone; R5020) treatment of 2D adherent cultures revealed functional PR signaling through MAPK-dependent p-PR as well as isoform specific expression of PR target genes encoding adhesion molecules (e.g. HEF1), cell cycle regulators (e.g. FOXO1), and glucocorticoid signaling proteins (e.g. CRISPLD2, NDRG1). Progestin treatment also dramatically inhibited proliferation which was recovered following growth factor stimulus, suggesting that PR signaling may promote cell quiescence (G0). To mimic dissemination of FTE cells from STICs, a 3D spheroid model was established. Interestingly, we observed that PR expression and activation greatly increased the formation, size and number of total spheroids compared to PR negative controls. Cells within these spheroids are predominantly Ki67-negative indicating that they are likely non-proliferative and potentially G0-arrested cells. When the spheroids were embedded into collagen, to mimic tumor cell invasion into the peritoneum, only PR+ spheroids exhibited invasive behavior. Additionally, PR-B+ spheroids were more invasive than PR-A+ spheroids; progestin treatment during 3D formation dramatically increased spheroid invasion. Notably, this is the opposite of our results in 2D cultures, where PR-A+ FTE cells display greater proliferative and migratory phenotypes relative to PR-B+ FTE cells. Taken together, our data suggest that activation of PR signaling promotes and enhances the formation and survival of non-adherent 3D FTE structures. Such effects of progesterone are predicted to influence the shedding, aggregation and dissemination of early STIC lesions that form ovarian and peritoneal metastases. Our findings demonstrate the importance of understanding the impact of steroid hormone receptors, including PR isoforms, on early ovarian cancer development.

A branching gene regulatory network dictating different aspects of a neuronal cell identity.

The nervous system displays a daunting cellular diversity. Neuronal subtypes differ from each other in several aspects, including their neurotransmitter expression and axon projection. These aspects can converge, but can also diverge, such that neurons expressing the same neurotransmitter may project axons to different targets. It is not well understood how regulatory programs converge/diverge to associate/dissociate different cell fate features. Studies of the Drosophila Tv1 neurons have identified a regulatory cascade, ladybird early→collier→apterous/eyes absent→dimmed, that specifies Tv1 neurotransmitter expression. Here, we conduct genetic and transcriptome analysis to address how other aspects of Tv1 cell fate are governed. We find that an initiator terminal selector gene triggers a feedforward loop that branches into different subroutines, each of which establishes different features of this one unique neuronal cell fate.

Epigenetic fates of gene silencing established by heterochromatin spreading in cell identity and genome stability.

Heterochromatin spreading, the propagation of repressive chromatin along the chromosome, is a reaction critical to genome stability and defense, as well as maintenance of unique cell fates. Here, we discuss the intrinsic properties of the spreading reaction and circumstances under which its products, formed distal to DNA-encoded nucleation sites, can be epigenetically maintained. Finally, we speculate that the epigenetic properties of heterochromatin evolved together with the need to stabilize cellular identity.

Enhanced Delta-Notch Lateral Inhibition Model Incorporating Intracellular Notch Heterogeneity and Tension-Dependent Rate of Delta-Notch Binding that Reproduces Sprouting Angiogenesis Patterns

Endothelial cells adopt unique cell fates during sprouting angiogenesis, differentiating into tip or stalk cells. The fate selection process is directed by Delta-Notch lateral inhibition pathway. Classical Delta-Notch models produce a spatial pattern of tip cells separated by a single stalk cell, or the salt-and-pepper pattern. However, classical models cannot explain alternative tip-stalk patterning, such as tip cells that are separated by two or more stalk cells. We show that lateral inhibition models involving only Delta and Notch proteins can also recapitulate experimental tip-stalk patterns by invoking two mechanisms, specifically, intracellular Notch heterogeneity and tension-dependent rate of Delta-Notch binding. We introduce our computational model and analysis where we establish that our enhanced Delta-Notch lateral inhibition model can recapitulate a greater variety of tip-stalk patterning which is previously not possible using classical lateral inhibition models. In our enhanced Delta-Notch lateral inhibition model, we observe the existence of a hybrid cell type displaying intermediate tip and stalk cells’ characteristics. We validate the existence of such hybrid cells by immuno-staining of endothelial cells with tip cell markers, Delta and CD34, which substantiates our enhanced model.

CD36 initiates the secretory phenotype during the establishment of cellular senescence.

Cellular senescence is a unique cell fate characterized by stable proliferative arrest and the extensive production and secretion of various inflammatory proteins, a phenomenon known as the senescence-associated secretory phenotype (SASP). The molecular mechanisms responsible for generating a SASP in response to senescent stimuli remain largely obscure. Here, using unbiased gene expression profiling, we discover that the scavenger receptor CD36 is rapidly upregulated in multiple cell types in response to replicative, oncogenic, and chemical senescent stimuli. Moreover, ectopic CD36 expression in dividing mammalian cells is sufficient to initiate the production of a large subset of the known SASP components via activation of canonical Src-p38-NF-κB signaling, resulting in the onset of a full senescent state. The secretome is further shown to be ligand-dependent, as amyloid-beta (Aβ) is sufficient to drive CD36-dependent NF-κB and SASP activation. Finally, loss-of-function experiments revealed a strict requirement for CD36 in secretory molecule production during conventional senescence reprogramming. Taken together, these results uncover the Aβ-CD36-NF-κB signaling axis as an important regulator of the senescent cell fate via induction of the SASP.

An Enhanced Delta-Notch Lateral Inhibition Model Incorporating Intracellular Notch Heterogenity and Tension-Dependent Rate of Delta-Notch Binding to Explain Cellular Patterning during Sprouting Angiogenesis

During sprouting angiogenesis, endothelial cells take on a spatial pattern of unique cell fates, becoming either the tip cell or the stalk cell. This process of fate selection is guided by lateral inhibition via the Delta-Notch signalling pathway. Classical Delta-Notch lateral inhibition models generate the salt-and-pepper pattern where tip cells are separated exactly by one stalk cell. However, it has been observed during angiogenesis that other forms of tip-stalk patterning are present, such as two or three stalk cells spaced between consecutive tip cells. To reproduce the diverse forms of angiogenic patterns, we develop a mathematical model that incorporates both intracellular Notch heterogeneity and tension-dependent rate of Delta-Notch binding into the classical lateral inhibition model. We first provide experimental evidence to justify the inclusion of these additional mechanisms. Next, we present our computational analysis where we demonstrate that unlike classical lateral inhibition models, our enhanced Delta-Notch lateral inhibition model is capable of explaining a larger range of cellular patterning during sprouting angiogenesis. A pivotal prediction in our enhanced Delta-Notch lateral inhibition model is the existence of intermediate cell states, i.e., cells that exhibit moderate Delta or Notch levels thus manifesting both tip cell and stalk cell traits. We provide experimental evidence that such intermediate cell states do exist by staining of endothelial cell cultures with CD34, a known tip cell marker, thus substantiating our enhanced model.

Epigenetic Plasticity Drives Adipogenic and Osteogenic Differentiation of Marrow-derived Mesenchymal Stem Cells

Terminal differentiation of multipotent stem cells is achieved through a coordinated cascade of activated transcription factors and epigenetic modifications that drive gene transcription responsible for unique cell fate. Within the mesenchymal lineage, factors such as RUNX2 and PPARγ are indispensable for osteogenesis and adipogenesis, respectively. We therefore investigated genomic binding of transcription factors and accompanying epigenetic modifications that occur during osteogenic and adipogenic differentiation of mouse bone marrow-derived mesenchymal stem cells (MSCs). As assessed by ChIP-sequencing and RNA-sequencing analyses, we found that genes vital for osteogenic identity were linked to RUNX2, C/EBPβ, retinoid X receptor, and vitamin D receptor binding sites, whereas adipocyte differentiation favored PPARγ, retinoid X receptor, C/EBPα, and C/EBPβ binding sites. Epigenetic marks were clear predictors of active differentiation loci as well as enhancer activities and selective gene expression. These marrow-derived MSCs displayed an epigenetic pattern that suggested a default preference for the osteogenic pathway; however, these patterns were rapidly altered near the Adipoq, Cidec, Fabp4, Lipe, Plin1, Pparg, and Cebpa genes during adipogenic differentiation. Surprisingly, we found that these cells also exhibited an epigenetic plasticity that enabled them to trans-differentiate from adipocytes to osteoblasts (and vice versa) after commitment, as assessed by staining, gene expression, and ChIP-quantitative PCR analysis. The osteogenic default pathway may be subverted during pathological conditions, leading to skeletal fragility and increased marrow adiposity during aging, estrogen deficiency, and skeletal unloading. Taken together, our data provide an increased mechanistic understanding of the epigenetic programs necessary for multipotent differentiation of MSCs that may prove beneficial in the development of therapeutic strategies.

FTOC-Based Analysis of Negative Selection.

Potentially harmful T cell precursors are removed from the conventional T cell pool by negative selection. This process can involve the induction of apoptosis, anergy, receptor editing or deviation into a regulatory T cell lineage. As such this process is essential for the health of an organism through its contribution to central and peripheral tolerance. While a great deal is known about the process, the precise mechanisms that regulate negative selection are not clear. Furthermore, the signals that distinguish the different forms of negative selection are not fully understood. Numerous models exist with the potential to address these questions in vitro and in vivo. This chapter describes methods of fetal thymic organ culture designed to analyze the signals that determine these unique cell fates.

In Vitro Analysis of Thymocyte Signaling.

From the moment a developing thymocyte expresses a TCR, it is subjected to numerous interactions with self-peptide/MHC complexes that determine its ultimate fate. These include death by neglect, negative selection (apoptosis and lineage deviation), positive selection, and lineage commitment. The identification of signals that govern these unique cell fates requires the ability to assess the activity, level of expression, subcellular location, and the molecular associations of numerous proteins within the developing T cell. Thus, this chapter describes methods designed to analyze thymocyte signaling under various types of peptide-based stimulation in vitro.

Tissue patterning and cellular mechanics.

In development, cells organize into biological tissues through cell growth, migration, and differentiation. Globally, this process is dictated by a genetically encoded program in which secreted morphogens and cell–cell interactions prompt the adoption of unique cell fates. Yet, at its lowest level, development is achieved through the modification of cell–cell adhesion and actomyosin-based contractility, which set the level of tension within cells and dictate how they pack together into tissues. The regulation of tension within individual cells and across large groups of cells is a major driving force of tissue organization and the basis of all cell shape change and cell movement in development.

The Drosophila T-box transcription factor Midline functions within the Notch–Delta signaling pathway to specify sensory organ precursor cell fates and regulates cell survival within the eye imaginal disc

We report that the T-box transcription factor Midline (Mid), an evolutionary conserved homolog of the vertebrate Tbx20 protein, functions within the Notch–Delta signaling pathway essential for specifying the fates of sensory organ precursor (SOP) cells. These findings complement an established history of research showing that Mid regulates the cell-fate specification of diverse cell types within the developing heart, epidermis and central nervous system. Tbx20 has been detected in unique neuronal and epithelial cells of embryonic eye tissues in both mice and humans. However, the mechanisms by which either Mid or Tbx20 function to regulate cell-fate specification or other critical aspects of eye development including cell survival have not yet been elucidated. We have also gathered preliminary evidence suggesting that Mid may play an indirect, but vital role in selecting SOP cells within the third-instar larval eye disc by regulating the expression of the proneural gene atonal. During subsequent pupal stages, Mid specifies SOP cell fates as a member of the Notch–Delta signaling hierarchy and is essential for maintaining cell viability by inhibiting apoptotic pathways. We present several new hypotheses that seek to understand the role of Mid in regulating developmental processes downstream of the Notch receptor that are critical for specifying unique cell fates, patterning the adult eye and maintaining cellular homeostasis during eye disc morphogenesis.

Spatially Dependent Dynamic MAPK Modulation by the Nde1-Lis1-Brap Complex Patterns Mammalian CNS

Regulating cell proliferation and differentiation in CNS development requires both extraordinary complexity and precision. Neural progenitors receive graded overlapping signals from midline signaling centers, yet each makes a unique cell fate decision in a spatiotemporally restricted pattern. The Nde1-Lis1 complex regulates individualized cell fate decisions based on the geographical location with respect to the midline. While cells distant from the midline fail to self-renew in the Nde1-Lis1 double-mutant CNS, cells embedded in the signaling centers showed marked overproliferation. A direct interaction between Lis1 and Brap, a mitogen-activated protein kinase (MAPK) signaling threshold modulator, mediates this differential response to mitogenic signal gradients. Nde1-Lis1 deficiency resulted in a spatially dependent alteration of MAPK scaffold Ksr and hyperactivation of MAPK. Epistasis analyses supported synergistic Brap and Lis1 functions. These results suggest that a molecular complex composed of Nde1, Lis1, and Brap regulates the dynamic MAPK signaling threshold in a spatially dependent fashion.

Continuous Expression of the Transcription Factor E2-2 Maintains the Cell Fate of Mature Plasmacytoid Dendritic Cells

The interferon-producing plasmacytoid dendritic cells (pDCs) share common progenitors with antigen-presenting classical dendritic cells (cDCs), yet they possess distinct morphology and molecular features resembling those of lymphocytes. It is unclear whether the unique cell fate of pDCs is actively maintained in the steady state. We report that the deletion of transcription factor E2-2 from mature peripheral pDCs caused their spontaneous differentiation into cells with cDC properties. This included the loss of pDC markers, increase in MHC class II expression and Tcell priming capacity, acquisition of dendritic morphology, and induction of cDC signature genes. Genome-wide chromatin immunoprecipitation revealed direct binding of E2-2 to key pDC-specific and lymphoid genes, as well as to certain genes enriched in cDCs. Thus, E2-2 actively maintains the cell fate of mature pDCs and opposes the "default" cDC fate, in part through direct regulation of lineage-specific gene expression programs.