Cell Fate Determination Research Articles

High-resolution profiling reveals coupled transcriptional and translational regulation of transgenes.

Concentrations of RNAs and proteins provide important determinants of cell fate. Robust gene circuit design requires an understanding of how the combined actions of individual genetic components influence both mRNA and protein levels. Here, we simultaneously measure mRNA and protein levels in single cells using HCR Flow-FISH for a set of commonly used synthetic promoters. We find that promoters generate differences in both the mRNA abundance and the effective translation rate of these transcripts. Stronger promoters not only transcribe more RNA but also show higher effective translation rates. While the strength of the promoter is largely preserved upon genome integration with identical elements, the choice of polyadenylation signal and coding sequence can generate large differences in the profiles of the mRNAs and proteins. We used long-read direct RNA sequencing to characterize full-length mRNA isoforms and observe remarkable uniformity of mRNA isoforms from the transgenic units. Together, our high-resolution profiling of transgenic mRNAs and proteins offers insight into the impact of common synthetic genetic components on transcriptional and translational mechanisms. By developing a novel framework for quantifying expression profiles of transgenes, we have established a system for comparing native and synthetic gene regulation and for building more robust transgenic systems.

Polycomb protein RYBP facilitates super-enhancer activity

BackgroundPolycomb proteins are conventionally known as global repressors in cell fate determination. However, recent observations have shown their involvement in transcriptional activation, the mechanisms of which need further investigation.MethodsHerein, multiple data from ChIP-seq, RNA-seq and HiChIP before or after RYBP depletion in embryonic stem cell (ESC), epidermal progenitor (EPC) and mesodermal cell (MEC) were analyzed.ResultsWe found that Polycomb protein RYBP occupies super-enhancer (SE) in ESCs, where core Polycomb group (PcG) components such as RING1B and EZH2 are minimally enriched. Depletion of RYBP results in impaired deposition of H3K27ac, decreased expression of SE-associated genes, and reducing the transcription of enhancer RNA at SE regions (seRNA). Regarding the mechanism of seRNA transcription, the Trithorax group (TrxG) component WDR5 co-localizes with RYBP at SEs, and is required for seRNA expression. RYBP depletion reduces WDR5 deposition at SE regions. In addition, TrxG-associated H3K4me3 tends to be enriched at SEs with high levels of seRNA transcription, and RYBP deficiency impairs the deposition of H3K4me3 at SEs. Structurally, RYBP is involved in both intra- and inter-SE interactions. Finally, RYBP generally localizes at SEs in both in vitro cell lines and in vivo tissue-derived cells, dysfunction of RYBP is associated with various cancers and developmental diseases.ConclusionRYBP cooperates with TrxG component to regulate SE activity. Dysfunction of RYBP relates to various diseases. The findings provide new insights into the transcriptionally active function of Polycomb protein in cell fate determination.

Zfp260 choreographs the early stage osteo-lineage commitment of skeletal stem cells

The initial fine-tuning processes are crucial for successful bone regeneration, as they guide skeletal stem cells through progenitor differentiation toward osteo- or chondrogenic fate. While fate determination processes are well-documented, the mechanisms preceding progenitor commitment remain poorly understood. Here, we identified a transcription factor, Zfp260, as pivotal for stem cell maturation into progenitors and directing osteogenic differentiation. Zfp260 is markedly up-regulated as cells transition from stem to progenitor stages; its dysfunction causes lineage arrest at the progenitor stage, impairing bone repair. Zfp260 is required for maintaining chromatin accessibility and regulates Runx2 expression by forming super-enhancer complexes. Furthermore, the PKCα kinase phosphorylates Zfp260 at residues Y173, S182, and S197, which are essential for its functional activity. Mutations at these residues significantly impair its functionality. These findings position Zfp260 as a vital factor bridging stem cell activation with progenitor cell fate determination, unveiling a element fundamental to successful bone regeneration.

WNT9A and WNT9B in Development and Disease

WNT9 paralogues, WNT9A and WNT9B, are secreted ligands driving both the canonical (β-catenin dependent) and non-canonical (β-catenin independent) Wnt signaling pathways. These pathways play roles in cell fate determination, embryonic patterning, bone development, and organogenesis, among other biological processes. Studies of Wnt9a and Wnt9b mutant animals demonstrate that they have specific and overlapping roles in these processes. Wnt9a is critical in directing stem and progenitor cell fate during hematopoietic stem cell development, proper bone formation, and chondrogenesis, while Wnt9b is important for kidney and heart development. Both proteins are essential in craniofacial development and convergent extension movements. Dysregulated expression of human WNT9A and WNT9B have been implicated in different cancers and disease, suggesting these proteins or their downstream pathways may represent potential therapeutic targets.

A multi-omic atlas of human embryonic skeletal development

Human embryonic bone and joint formation is determined by coordinated differentiation of progenitors in the nascent skeleton. The cell states, epigenetic processes and key regulatory factors that underlie lineage commitment of these cells remain elusive. Here we applied paired transcriptional and epigenetic profiling of approximately 336,000 nucleus droplets and spatial transcriptomics to establish a multi-omic atlas of human embryonic joint and cranium development between 5 and 11 weeks after conception. Using combined modelling of transcriptional and epigenetic data, we characterized regionally distinct limb and cranial osteoprogenitor trajectories across the embryonic skeleton and further described regulatory networks that govern intramembranous and endochondral ossification. Spatial localization of cell clusters in our in situ sequencing data using a new tool, ISS-Patcher, revealed mechanisms of progenitor zonation during bone and joint formation. Through trajectory analysis, we predicted potential non-canonical cellular origins for human chondrocytes from Schwann cells. We also introduce SNP2Cell, a tool to link cell-type-specific regulatory networks to polygenic traits such as osteoarthritis. Using osteolineage trajectories characterized here, we simulated in silico perturbations of genes that cause monogenic craniosynostosis and implicate potential cell states and disease mechanisms. This work forms a detailed and dynamic regulatory atlas of bone and cartilage maturation and advances our fundamental understanding of cell-fate determination in human skeletal development.

Frizzled5 controls murine intestinal epithelial cell plasticity through organization of chromatin accessibility.

The homeostasis of the intestinal epithelium relies on intricate yet insufficiently understood mechanisms of intestinal epithelial plasticity. Here, we elucidate the pivotal role of Frizzled5 (Fzd5), a Wnt pathway receptor, as a determinant of murine intestinal epithelial cell fate. Deletion of Fzd5 in Lgr5+ intestinal stem cells (ISCs) impairs their self-renewal, whereas its deletion in Krt19+ cells disrupts lineage generation, without affecting crypt integrity in either case. However, a broader deletion of Fzd5 across the epithelium leads to substantial crypt deterioration. Integrated analysis of single-cell RNA sequencing (scRNA-seq) and single-cell ATAC-seq (scATAC-seq) identifies that Fzd5 governs chromatin accessibility, orchestrating the regulation of stem- and lineage-related gene expression mainly in ISCs and progenitor cells. In summary, our findings provide insights into the regulatory role of Fzd5 in governing intestinal epithelial plasticity.

The people behind the papers - Maarten Bebelman, Lenka Belicova and Marino Zerial.

Hepatoblasts are the bipotential progenitors of liver cells that differentiate into either hepatocytes or cholangiocytes. In their study, Marino Zerial and colleagues reveal how hepatoblast polarisation and lumen morphogenesis are linked to hepatocyte cell fate determination. To know more about their work, we spoke to the first authors, Maarten Bebelman and Lenka Belicova, and the corresponding author, Marino Zerial, Director and Scientific Member at the Max Planck Institute of Molecular Cell Biology and Genetics.

JAK/STAT signaling maintains an intermediate cell population during prostate basal cell fate determination.

Unipotent basal and luminal stem cells maintain prostate homeostasis, with an intermediate cell population emerging during prostate inflammation or cancer. However, the identities of basal stem cell and intermediate cell population remain unclear. Here we identified a rare intermediate cell population expressing luminal markers (termed Basal-B) with enhanced organoid formation capacity, and a larger basal population (termed Basal-A). Genetic lineage tracing revealed Basal-B cells represented a transient basal stem cell state during prostate homeostasis and androgen-mediated regeneration. Activated JAK/STAT signaling was identified in Basal-B cells, and its inhibition significantly reduced Basal-B markers expression. Inflammation increased Basal-B-to-luminal cell transdifferentiation, but JAK/STAT inhibition notably attenuated this effect. Pten gene deletion increased Nkx3.1-expressing Basal-B-like cell population and led to neoplasia. In humans, h-Basal-B cells were more prevalent in benign prostate hyperplasia. This study reveals the identities of intermediate Basal-B cells and underscores the role of JAK/STAT signaling in prostate cell fate determination.

Optimisation of cell fate determination for cultivated muscle differentiation

Production of cultivated meat requires defined medium formulations for the robust differentiation of myogenic cells into mature skeletal muscle fibres in vitro. Although these formulations can drive myogenic differentiation levels comparable to serum-starvation-based protocols, the resulting cultures are often heterogeneous, with a significant proportion of cells not participating in myofusion, limiting maturation of the muscle. To address this problem, we employed RNA sequencing to analyse heterogeneity in differentiating bovine satellite cells at single-nucleus resolution, identifying distinct cellular subpopulations including proliferative cells that fail to exit the cell cycle and quiescent ‘reserve cells’ that do not commit to myogenic differentiation. Our findings indicate that the MEK/ERK, NOTCH, and RXR pathways are active during the initial stages of myogenic cell fate determination, and by targeting these pathways, we can promote cell cycle exit while reducing reserve cell formation. This optimised medium formulation consistently yields fusion indices close to 100% in 2D culture. Furthermore, we demonstrate that these conditions enhance myotube formation and actomyosin accumulation in 3D bovine skeletal muscle constructs, providing proof of principle for the generation of highly differentiated cultivated muscle with excellent mimicry to traditional muscle.

Adeno-Associated Virus-Mediated Dickkopf-1 Gene Transduction Reduces Silica-Induced Oxidative Stress and Silicosis in Mouse Lung.

Aims: Silicosis is a lung disease caused by inhalation of silica particles. Both silica-induced oxidative stress and aberrant activation of the Wnt/β-catenin signaling pathway are potential targets in the treatment of pulmonary fibrosis. Dickkopf-1 (Dkk1), an inhibitor of the Wnt/β-catenin signaling pathway, plays regulatory roles in cell fate determination and immune responses. Our previous study demonstrated that adenoviral vector-mediated Dkk1 gene transfer alleviated the silica-induced mouse silicosis. However, the mechanism of therapeutic action of Dkk1 in silicosis is yet completely understood; together with the drawbacks of adenoviral vectors in gene therapy, we investigated the therapeutic effect and mechanisms of Dkk1 by employing an adeno-associated virus (AAV) vector in a silicosis mouse model. Results: The AAV vector could efficiently transduce the Dkk1 gene in silicotic lung during both the early and the late phases of disease, resulting in an alleviation of silicotic lesions, improvement of pulmonary compliance, and radiological findings. Mechanistic studies further demonstrated that the transduction of Dkk1 inhibited the silica-activated Wnt/β-catenin signaling and reduced the silica-induced reactive oxygen species-producing enzyme NADPH oxidase 4, oxidative stress regulator nuclear factor erythroid 2-related factor 2, and signaling molecules binding immunoglobulin protein and C/EBP homologous protein. In addition, shRNA-mediated downregulation of Dkk1 exacerbated the progression of silicosis in mice, whereas the treatment of ROS scavenger n-acetylcysteine showed a comparable mitigation of silicosis that was seen in the AAV-Dkk1 treatment. Innovation and Conclusion: This study provides an insight into the mechanism by which Dkk1 inhibits the silica-induced Wnt signaling and oxidative stress to mitigate the pathogenesis of lung silicosis and evidence of the potential of AAV-mediated Dkk1 gene transfer as an alternative approach in silicosis treatment. Antioxid. Redox Signal. 00, 000-000.

Delineating Notch1 and Notch2: Receptor-Specific Significance and Therapeutic Importance of Pinpoint Targeting Strategies for Hematological Malignancies.

Notch1 and Notch2, transmembrane receptors belonging to the Notch family, are pivotal mediators of intercellular communication and have profound implications including cell fate determination, embryonic development, and tissue homeostasis in various cellular processes. Despite their structural homology, Notch1 and Notch2 exhibit discrete phenotypic characteristics and functional nuances that necessitate their individualized targeting in specific medical scenarios. Aberrant Notch signaling, often driven by the dysregulated activity of one receptor over the other, is implicated under various pathological conditions. Notch1 dysregulation is frequently associated with T-cell acute lymphoblastic leukemia, whereas Notch2 perturbations are linked to B-cell malignancies and solid tumors, including breast cancer. Hence, tailored therapeutic interventions that selectively inhibit the relevant Notch receptor need to be devised to disrupt the signaling pathways driving the specific disease phenotype. In this review, we emphasize the importance of distinct tissue-specific expression patterns, functional divergence, disease-specific considerations, and the necessity to minimize off-target effects that collectively underscore the significance of "individualized" targeting for Notch1 and Notch2. This comprehensive review sheds light on the receptor-specific characteristics of Notch1 and Notch2, providing insights into their roles in cellular processes and offering opportunities for developing tailored therapeutic interventions in the fields of biomedical research and clinical practice.

TMIC-77. ELUCIDATING THE ROLE OF PTPRZ1 SIGNALING IN DRIVING GLIOBLASTOMA PROGRESSION USING THE NOVEL HUMAN ORGANOID TUMOR TRANSPLANTATION SYSTEM

Abstract Glioblastoma (GBM), the most malignant and aggressive primary brain tumor, is driven by both intrinsic genetic mutations and extrinsic factors from the tumor microenvironment (TME). GBM manipulates the TME to enhance its survival and proliferation through mechanisms such as forming functional synapses with adjacent normal cells and secreting immunosuppressive factors. Our laboratory has developed a novel human organoid tumor transplantation (HOTT) model, which allows for the interrogation of tumor-normal cell crosstalk at a molecular level and facilitates the dissection of specific signaling pathways that drive tumor progression and maintenance. Using single-cell transcriptomics on human GBM HOTTs, we identified the PTN-PTPRZ1 ligand-receptor interaction as a critical mediator of tumor-microenvironment communication. PTPRZ1, a receptor tyrosine phosphatase, has been previously implicated in glioma cell migration, and our data further elucidate its role in modulating tumor behavior. Specifically, we demonstrated that knockdown of PTPRZ1 within the TME of human cortical organoids significantly enhances glioma cell migration and inhibits differentiation, underscoring the essential role of PTPRZ1 in maintaining the tumor’s invasive phenotype. Further pharmacological inhibition of PTPRZ1’s catalytic activity with a small molecule revealed that these effects on cell fate determination are independent of its phosphatase activity. This suggests that PTPRZ1 exerts critical non-catalytic functions that are pivotal for GBM progression. To further investigate these non-catalytic mechanisms, we propose utilizing single-cell transcriptomics coupled with proximity labeling and proteomics. This approach will allow us to comprehensively map PTPRZ1-mediated signaling networks and elucidate their impact on tumor biology, potentially unveiling novel therapeutic targets. Our findings revealed a novel axis of GBM biology mediated by PTPRZ1 and highlighted the importance of considering microenvironmental interactions in the development of effective therapeutics for GBM.

Characterising Cancer Cell Responses to Cyclic Hypoxia Using Mathematical Modelling

In vivo observations show that oxygen levels in tumours can fluctuate on fast and slow timescales. As a result, cancer cells can be periodically exposed to pathologically low oxygen levels; a phenomenon known as cyclic hypoxia. Yet, little is known about the response and adaptation of cancer cells to cyclic, rather than, constant hypoxia. Further, existing in vitro models of cyclic hypoxia fail to capture the complex and heterogeneous oxygen dynamics of tumours growing in vivo. Mathematical models can help to overcome current experimental limitations and, in so doing, offer new insights into the biology of tumour cyclic hypoxia by predicting cell responses to a wide range of cyclic dynamics. We develop an individual-based model to investigate how cell cycle progression and cell fate determination of cancer cells are altered following exposure to cyclic hypoxia. Our model can simulate standard in vitro experiments, such as clonogenic assays and cell cycle experiments, allowing for efficient screening of cell responses under a wide range of cyclic hypoxia conditions. Simulation results show that the same cell line can exhibit markedly different responses to cyclic hypoxia depending on the dynamics of the oxygen fluctuations. We also use our model to investigate the impact of changes to cell cycle checkpoint activation and damage repair on cell responses to cyclic hypoxia. Our simulations suggest that cyclic hypoxia can promote heterogeneity in cellular damage repair activity within vascular tumours.

Unraveling the Role of Wnt Signaling Pathway in the Pathogenesis of Autism Spectrum Disorder (ASD): A Systematic Review.

Autism spectrum disorder (ASD), or simply autism, is a neurodevelopmental disorder characterized by social communication deficit, restricted interests, and repetitive behavior. Several studies suggested a link between autism and the dysregulation of the Wnt signaling pathway which is mainly involved in cell fate determination, cell migration, cell polarity, neural patterning, and organogenesis. Despite the absence of effective therapy, significant progress has been made in understanding the pathogenesis of ASD. Neuropharmacological studies showed that drugs acting on the Wnt pathway like Canagliflozin can alleviate autistic-like behavior in animal models. Hence, this pathway could potentially be a futuristic therapeutic target to mitigate autism's symptoms. This systematic review aims to collect and analyze evidence that elucidates how alterations in the Wnt pathway may contribute to the pathogenesis of autism in animal models at the molecular, cellular, and physiological levels. Comprehensive searches were conducted across multiple databases, including PubMed, Web of Science, Embase, and Scopus to identify relevant studies up to March 2024. The inclusion criteria encompassed experimental studies that focused on the link between autism and this pathway, and the quality assessment was ensured by SYRCLE's risk of bias tools. Collectively, the included articles highlighted the possible implication of this pathway in the abnormalities found in autism, which impacted processes such as energy metabolism, oxidative stress, and neurogenesis. These alterations could underlie autistic behavior by affecting synaptic transmission and mitochondrial function.

Bmgsb is involved in the determination of cell fate by affecting the cell cycle genes in the silk gland of Bombyx mori

Silk gland is the only organ of silkworm that can produce silk protein, which is a natural macromolecular protein complex and widely utilized in various fields such as biomaterials and biomedicine. The development of silk gland and the expression patterns of silk protein crucial for the silk industry. In this study, the function of a transcription factor Bmgsb was investigated with CRISPR/Cas9 and transgenic system. It was found that the homozygous individuals in the Bmgsb KO line experienced spinning failure and pupae death, the AMSG exhibited defects, and the ASG displayed abnormal curvature. These phenotypes were accompanied by increased DNA endoreplication and significantly upregulated expression of fibroin genes in the ASG. RT-qPCR results confirmed significant upregulation of cell cycle-related genes, including cyclin G and cyclin T in the Bmgsb KO line. Furthermore, ectopic expression of Bmgsb in the PSG weakened PSG curvature, inhibited DNA endoreplication, and downregulated the expression of fibroin genes. These findings strongly suggest that Bmgsb plays a crucial role in determining cell fate in the silk gland and regulating the expression of silk protein through the cyclin pathway. Our research provides a theoretical foundation for further studies on organ differentiation and have implications for the silk industry.

ScLTdb: a comprehensive single-cell lineage tracing database.

Single-cell lineage tracing (scLT) is a powerful technique that integrates cellular barcoding with single-cell sequencing technologies. This new approach enables the simultaneous measurement of cell fate and molecular profiles at single-cell resolution, uncovering the gene regulatory program of cell fate determination. However, a comprehensive scLT database is not yet available. Here, we present the single-cell lineage tracing database (scLTdb, https://scltdb.com) containing 109 datasets that are manually curated and analyzed through a standard pipeline. The scLTdb provides interactive analysis modules for visualizing and re-analyzing scLT datasets, especially the comprehensive cell fate analysis and lineage relationship analysis. Importantly, scLTdb also allows users to identify fate-related gene signatures. In conclusion, scLTdb provides an interactive interface of scLT data exploration and analysis, and will facilitate the understanding of cell fate decision and lineage commitment in development and diseases.

Unlocking the secrets of Cardiac development and function: the critical role of FHL2.

FHL2 (Four-and-a-half LIM domain protein 2) is a crucial factor involved in cardiac morphogenesis, the process by which the heart develops its complex structure. It is expressed in various tissues during embryonic development, including the developing heart, and has been shown to play important roles in cell proliferation, differentiation, and migration. FHL2 interacts with multiple proteins to regulate cardiac development as a coactivator or a corepressor. It is involved in cardiac specification and determination of cell fate, cardiomyocyte growth, cardiac remodeling, myofibrillogenesis, and the regulation of HERG channels. Targeting FHL2 has therapeutic implications as it could improve cardiac function, control arrhythmias, alleviate heart failure, and maintain cardiac integrity in various pathological conditions. The identification of FHL2 as a signature gene in atrial fibrillation suggests its potential as a diagnostic marker and therapeutic target for this common arrhythmia.

Ependymal cell lineage reprogramming as a potential therapeutic intervention for hydrocephalus

Hydrocephalus is a common neurological condition, characterized by the excessive accumulation of cerebrospinal fluid in the cerebral ventricles. Primary treatments for hydrocephalus mainly involve neurosurgical cerebrospinal fluid diversion, which hold high morbidity and failure rates, highlighting the necessity for the discovery of novel therapeutic approaches. Although the pathophysiology of hydrocephalus is highly multifactorial, impaired function of the brain ependymal cells plays a fundamental role in hydrocephalus. Here we show that GemC1 and McIdas, key regulators of multiciliated ependymal cell fate determination, induce direct cellular reprogramming towards ependyma. Our study reveals that ectopic expression of GemC1 and McIdas reprograms cortical astrocytes and programs mouse embryonic stem cells into ependyma. McIdas is sufficient to establish functional activity in the reprogrammed astrocytes. Furthermore, we show that McIdas’ expression promotes ependymal cell regeneration in two different postnatal hydrocephalus mouse models: an intracranial hemorrhage and a genetic form of hydrocephalus and ameliorates the cytoarchitecture of the neurogenic niche. Our study provides evidence on the restoration of ependyma in animal models mimicking hydrocephalus that could be exploited towards future therapeutic interventions.

Negative feedback regulation of GLABRA1 contributes to epidermal cell patterning in the Arabidopsis root

GLABRA1 (GL1), which encodes an R2R3 MYB transcription factor, is a key regulator of trichome patterning in the aerial organs of Arabidopsis (Arabidopsis thaliana). Although it has been generally assumed that GL1 functions exclusively in shoots and is not expressed in roots, reverse transcription polymerase chain reaction (RT-PCR) analysis has revealed that GL1 is indeed expressed in roots. To investigate whether GL1 plays a role in root epidermal patterning, we analyzed the effects of gl1 mutations in sensitized genetic backgrounds. Our findings show that gl1 mutants enhance the root epidermal phenotype of a weak allele of the werewolf (wer) mutant and suppress the phenotype of the caprice (cpc) mutant. We also demonstrate that the GL1 promoter is active in N-position epidermal cells, and that the GFP-GL1 fusion protein is predominantly localized in the nucleus of N-position cells. Furthermore, we provide evidence that GL1 expression is positively regulated by WER, GLABRA3, ENHANCER OF GLABRA3, and TRANSPARENT TESTA GLABRA1, while negatively regulated by CPC, TRIPTYCHON, and GLABRA2 (GL2). Notably, GL2, which is positively regulated by GL1, moderately represses GL1 expression, and both GL1 and GL2 are positively regulated by WER in N-position cells. These findings suggest that a negative feedback regulation of GL1 expression via GL2 contributes to the fine-tuning of non-hair cell fate determination in Arabidopsis root epidermis.

Palmitoylated Importin α Regulates Mitotic Spindle Orientation Through Interaction with NuMA.

Regulation of cell division orientation is a fundamental process critical to differentiation and tissue homeostasis. Microtubules emanating from the mitotic spindle pole bind a conserved complex of proteins at the cell cortex which orients the spindle and ultimately the cell division plane. Control of spindle orientation is of particular importance in developing tissues, such as the developing brain. Misorientation of the mitotic spindle and thus subsequent division plane misalignment can contribute to improper segregation of cell fate determinants in developing neuroblasts, leading to a rare neurological disorder known as microcephaly. We demonstrate that the nuclear transport protein importin α, when palmitoylated, plays a critical role in mitotic spindle orientation through localizing factors, such as NuMA, to the cell cortex. We also observe craniofacial developmental defects in Xenopus laevis when importin α palmitoylation is abrogated, including smaller head and brains, a hallmark of spindle misorientation and microcephaly. These findings characterize not only a role for importin α in spindle orientation, but also a broader role for importin α palmitoylation which has significance for many cellular processes.