Genetic Perturbations Research Articles

NEMF mutations in mice illustrate how Importin-β specific nuclear transport defects recapitulate neurodegenerative disease hallmarks.

Pathological disruption of Nucleocytoplasmic Transport (NCT), such as the mis-localization of nuclear pore complex proteins (Nups), nuclear transport receptors, Ran-GTPase, and RanGAP1, are seen in both animal models and in familial and sporadic forms of amyotrophic lateral sclerosis (ALS), frontal temporal dementia and frontal temporal lobar degeneration (FTD\FTLD), and Alzheimer's and Alzheimer's Related Dementias (AD/ADRD). However, the question of whether these alterations represent a primary cause, or a downstream consequence of disease is unclear, and what upstream factors may account for these defects are unknown. Here, we report four key findings that shed light on the upstream causal role of Importin-β-specific nuclear transport defects in disease onset. First, taking advantage of two novel mouse models of NEMF neurodegeneration (NemfR86S and NemfR487G) that recapitulate many cellular and biochemical aspects of neurodegenerative diseases, we find an Importin-β-specific nuclear import block. Second, we observe cytoplasmic mis-localization and aggregation of multiple proteins implicated in the pathogenesis of ALS/FTD and AD/ADRD, including TDP43, Importin-β, RanGap1, and Ran. These findings are further supported by a pathological interaction between Importin-β and the mutant NEMFR86S protein in cytoplasmic accumulations. Third, we identify similar transcriptional dysregulation in key genes associated with neurodegenerative disease. Lastly, we show that even transient pharmaceutical inhibition of Importin-β in both mouse and human neuronal and non-neuronal cells induces key proteinopathies and transcriptional alterations seen in our mouse models and in neurodegeneration. Our convergent results between mouse and human neuronal and non-neuronal cellular biology provide mechanistic evidence that many of the mis-localized proteins and dysregulated transcriptional events seen in multiple neurodegenerative diseases may in fact arise primarily from a primary upstream defect in Importin- β nuclear import. These findings have critical implications for investigating how sporadic forms of neurodegeneration may arise from presently unidentified genetic and environmental perturbations in Importin-β function.

DRADTiP: Drug repurposing for aging disease through drug-target interaction prediction

MotivationThe greatest risk factor for many non-communicable diseases is aging. Studies on model organisms have demonstrated that genetic and chemical perturbation alterations can lengthen longevity and overall health. However, finding longevity-enhancing medications and their related targets is difficult. MethodIn this work, we designed a novel drug repurposing model by identifying the interaction between aging-related genes or targets and drugs similar to aging disease. Each disease is associated with certain specific genetic factors for the occurrence of that disease. The factors include gene expression, pathway, miRNA, and degree of genes in the protein-protein interaction network. In this paper, we aim to find the drugs that prolong the life span of humans with their aging-related targets using the above-mentioned factors. In addition, the contribution or importance of each factor may vary among drugs and targets. Therefore, we designed a novel multi-layer random walk-based network representation learning model including node and edge weight to learn the features of drugs and targets respectively. ResultThe performance of the proposed model is demonstrated using k-fold cross-validation (k = 5). This model achieved better performance with scores of 0.93 and 0.91 for precision and recall respectively. The drugs identified by the system are evaluated to be potential candidates for aging since the degree of interaction between the potential drugs and their gene sets are high. In addition, the genes that are interacting with drugs produce the same biological functions. Hence the life span of the human will be increased or prolonged.

DRMY1 promotes robust morphogenesis in Arabidopsis by sustaining the translation of cytokinin-signaling inhibitor proteins.

Robustness is the invariant development of phenotype despite environmental changes and genetic perturbations. In the Arabidopsis flower bud, four sepals robustly initiate and grow to a constant size to enclose and protect the inner floral organs. We previously characterized the mutant development-related myb-like 1 (drmy1), where 3-5 sepals initiate variably and grow to different sizes, compromising their protective function. The molecular mechanism underlying this loss of robustness was unclear. Here, we show that drmy1 has reduced TARGET OF RAPAMYCIN (TOR) activity, ribosomal content, and translation. Translation reduction decreases the protein level of ARABIDOPSIS RESPONSE REGULATOR7 (ARR7) and ARABIDOPSIS HISTIDINE PHOSPHOTRANSFER PROTEIN 6 (AHP6), two cytokinin-signaling inhibitors that are normally rapidly produced before sepal initiation. The resultant upregulation of cytokinin signaling disrupts robust auxin patterning and sepal initiation. Our work shows that the homeostasis of translation, a ubiquitous cellular process, is crucial for the robust spatiotemporal patterning of organogenesis.

Wnt/β-Catenin Signaling Regulates Yap/Taz Activity during Embryonic Development in Zebrafish.

Hippo-YAP/TAZ and Wnt/β-catenin signaling pathways, by controlling proliferation, migration, cell fate, stemness, and apoptosis, are crucial regulators of development and tissue homeostasis. We employed zebrafish embryos as a model system to elucidate in living reporter organisms the crosstalk between the two signaling pathways. Co-expression analysis between the Wnt/β-catenin Tg(7xTCF-Xla.Siam:GFP)ia4 and the Hippo-Yap/Taz Tg(Hsa.CTGF:nlsmCherry)ia49 zebrafish reporter lines revealed shared spatiotemporal expression profiles. These patterns were particularly evident in key developmental regions such as the midbrain-hindbrain boundary (MHB), epidermis, muscles, neural tube, notochord, floorplate, and otic vesicle. To investigate the relationship between the Wnt/β-catenin pathway and Hippo-Yap/Taz signaling in vivo, we conducted a series of experiments employing both pharmacological and genetic strategies. Modulation of the Wnt/β-catenin pathway with IWR-1, XAV939, or BIO resulted in a significant regulation of the Yap/Taz reporter signal, highlighting a clear correlation between β-catenin and Yap/Taz activities. Furthermore, genetic perturbation of the Wnt/β-catenin pathway, by APC inhibition or DKK1 upregulation, elicited evident and robust alteration of Yap/Taz activity. These findings revealed the intricate regulatory mechanisms underlying the crosstalk between the Wnt/β-catenin and Hippo-Yap/Taz signaling, shedding light on their roles in orchestrating developmental processes in vivo.

Leukemia-derived apelin selects endothelial niche clones to promote tumorigenesis.

Hematopoietic stem cells are regulated by endothelial and mesenchymal stromal cells in the marrow niche1-3. Leukemogenesis was long believed to be solely driven by genetic perturbations in hematopoietic cells but introduction of genetic mutations in the microenvironment demonstrated the ability of niche cells to drive disease progression4-8. The mechanisms by which the stem cell niche induces leukemia remain poorly understood. Here, using cellular barcoding in zebrafish, we found that clones of niche endothelial and stromal cells are significantly expanded in leukemic marrows. The pro-angiogenic peptide apelin secreted by leukemic cells induced sinusoidal endothelial cell clonal selection and transcriptional reprogramming towards an angiogenic state to promote leukemogenesis in vivo. Overexpression of apelin in normal hematopoietic stem cells led to clonal amplification of the niche endothelial cells and promotes clonal dominance of blood cells. Knock-out of apelin in leukemic zebrafish resulted in a significant reduction in disease progression. Our results demonstrate that leukemic cells remodel the clonal and transcriptional landscape of the marrow niche to promote leukemogenesis and provide a potential therapeutic opportunity for anti-apelin treatment.

MEK Inhibitors Lead to PDGFR Pathway Upregulation and Sensitize Tumors to RAF Dimer Inhibitors in NF1-Deficient Malignant Peripheral Nerve Sheath Tumor.

Malignant peripheral nerve sheath tumor (MPNST) is a highly aggressive subtype of soft-tissue sarcoma with a high propensity to metastasize and extremely limited treatment options. Loss of the RAS-GAP NF1 leads to sustained RAF/MEK/ERK signaling in MPNST. However, single-agent MEK inhibitors (MEKi) have failed to elicit a sustained inhibition of the MAPK signaling pathway in MPNST. We used pharmacological, biochemical, and genetic perturbations of the receptor tyrosine kinase and MAPK signaling pathway regulators to investigate the mechanisms of MEKi resistance and evaluated combination therapeutic strategies in various preclinical MPNST models in vitro and in vivo. Here, we report that MEKi treatment resistance in MPNST involves two adaptive pathways: direct transcriptional upregulation of the receptor tyrosine kinase PDGFRβ and MEKi-induced increase in RAF dimer formation and activation of downstream signaling. Although the pharmacologic combination of a MEKi with a PDGFRβ-specific inhibitor was more effective than treatment with the MEKi alone, the combination of the MEKi and RAF dimer inhibitors led to a robust inhibition of MAPK pathway signaling. This combination treatment was effective in vitro and in vivo, as demonstrated by the significant increase in drug synergism and its high effectiveness in decreasing MPNST viability. Our findings suggest that the combination of MEKis and PDGFR and/or RAF dimer inhibitors can overcome MEKi resistance and may serve as a novel targeted therapeutic strategy for patients with NF1-deficient MPNST, which in turn could impact future clinical investigations for this patient population.

Endothelial cell elongation and alignment in response to shear stress requires acetylation of microtubules.

The innermost layer of the vessel wall is constantly subjected to recurring and relenting mechanical forces by virtue of their direct contact with blood flow. Endothelial cells of the vessel are exposed to distension, pressure, and shear stress; adaptation to these hemodynamic forces requires significant remodeling of the cytoskeleton which includes changes in actin, intermediate filaments, and microtubules. While much is known about the effect of shear stress on the endothelial actin cytoskeleton; the impact of hemodynamic forces on the microtubule network has not been investigated in depth. Here we used imaging techniques and protein expression analysis to characterize how pharmacological and genetic perturbations of microtubule properties alter endothelial responses to laminar shear stress. Our findings revealed that pharmacological suppression of microtubule dynamics blocked two typical responses to laminar shear stress: endothelial elongation and alignment. The findings demonstrate the essential contribution of the microtubule network to changes in cell shape driven by mechanical forces. Furthermore, we observed a flow-dependent increase in microtubule acetylation that occurred early in the process of cell elongation. Pharmacological manipulation of microtubule acetylation showed a direct and causal relationship between acetylation and endothelial elongation. Finally, genetic inactivation of aTAT1, a microtubule acetylase, led to significant loss of acetylation as well as inhibition of cell elongation in response to flow. In contrast, loss of HDAC6, a microtubule deacetylase, resulted in robust microtubule acetylation with cells displaying faster kinetics of elongation and alignment. Taken together, our findings uncovered the critical contributions of HDAC6 and aTAT1, that through their roles in the regulation of microtubule acetylation, are key mediators of endothelial mechanotransduction.

Flynotyper 2.0: an updated tool for rapid quantitative assessment of Drosophila eye phenotypes.

About two-thirds of the genes in the Drosophila melanogaster genome are also involved in its eye development, making the Drosophila eye an ideal system for genetic studies. We previously developed Flynotyper, a software that uses image processing operations to identify and quantify the degree of roughness by measuring disorderliness of ommatidial arrangement in the fly eye. This software has enabled researchers to quantify morphological defects of thousands of eye images caused by genetic perturbations. Here, we present Flynotyper 2.0, a software that has an updated computer vision library, improved performance, and a streamlined pipeline for high-throughput analysis of multiple eye images. We also tested several batches of Drosophila eye images to ensure robustness and reproducibility of the updated Flynotyper 2.0 software.

CRISPRi with barcoded expression reporters dissects regulatory networks in human cells.

Genome-wide CRISPR screens have emerged as powerful tools for uncovering the genetic underpinnings of diverse biological processes. Incisive screens often depend on directly measuring molecular phenotypes, such as regulated gene expression changes, provoked by CRISPR-mediated genetic perturbations. Here, we provide quantitative measurements of transcriptional responses in human cells across genome-scale perturbation libraries by coupling CRISPR interference (CRISPRi) with barcoded expression reporter sequencing (CiBER-seq). To enable CiBER-seq in mammalian cells, we optimize the integration of highly complex, barcoded sgRNA libraries into a defined genomic context. CiBER-seq profiling of a nuclear factor kappa B (NF-κB) reporter delineates the canonical signaling cascade linking the transmembrane TNF-alpha receptor to inflammatory gene activation and highlights cell-type-specific factors in this response. Importantly, CiBER-seq relies solely on bulk RNA sequencing to capture the regulatory circuit driving this rapid transcriptional response. Our work demonstrates the accuracy of CiBER-seq and its potential for dissecting genetic networks in mammalian cells with superior time resolution.

Signaling proteins in HSC fate determination are unequally segregated during asymmetric cell division.

Hematopoietic stem cells (HSCs) continuously replenish mature blood cells with limited lifespans. To maintain the HSC compartment while ensuring output of differentiated cells, HSCs undergo asymmetric cell division (ACD), generating two daughter cells with different fates: one will proliferate and give rise to the differentiated cells' progeny, and one will return to quiescence to maintain the HSC compartment. A balance between MEK/ERK and mTORC1 pathways is needed to ensure HSC homeostasis. Here, we show that activation of these pathways is spatially segregated in premitotic HSCs and unequally inherited during ACD. A combination of genetic and chemical perturbations shows that an ERK-dependent mechanism determines the balance between pathways affecting polarity, proliferation, and metabolism, and thus determines the frequency of asymmetrically dividing HSCs. Our data identify druggable targets that modulate HSC fate determination at the level of asymmetric division.

Learning meaningful representation of single-neuron morphology via large-scale pre-training.

Single-neuron morphology, the study of the structure, form, and shape of a group of specialized cells in the nervous system, is of vital importance to define the type of neurons, assess changes in neuronal development and aging and determine the effects of brain disorders and treatments. Despite the recent surge in the amount of available neuron morphology reconstructions due to advancements in microscopy imaging, existing computational and deep learning methods for modeling neuron morphology have been limited in both scale and accuracy. In this paper, we propose MorphRep, a model for learning meaningful representation of neuron morphology pre-trained with over 250 000 existing neuron morphology data. By encoding the neuron morphology into graph-structured data, using graph transformers for feature encoding and enforcing the consistency between multiple augmented views of neuron morphology, MorphRep achieves the state of the art performance on widely used benchmarking datasets. Meanwhile, MorphRep can accurately characterize the neuron morphology space across neuron morphometrics, fine-grained cell types, brain regions and ages. Furthermore, MorphRep can be applied to distinguish neurons under a wide range of conditions, including genetic perturbation, drug injection, environment change and disease. In summary, MorphRep provides an effective strategy to embed and represent neuron morphology and can be a valuable tool in integrating cell morphology into single-cell multiomics analysis. The codebase has been deposited in https://github.com/YaxuanLi-cn/MorphRep.

Genetic Regulatory Perturbation of Gene Expression Impacted by Genomic Introgression in Fiber Development of Allotetraploid Cotton.

Interspecific genomic introgression is an important evolutionary process with respect to the generation of novel phenotypic diversity and adaptation. A key question is how gene flow perturbs gene expression networks and regulatory interactions. Here, an introgression population of two species of allopolyploid cotton (Gossypium) to delineate the regulatory perturbations of gene expression regarding fiber development accompanying fiber quality change is utilized. De novo assembly of the recipient parent (G. hirsutum Emian22) genome allowed the identification of genomic variation and introgression segments (ISs) in 323 introgression lines (ILs) from the donor parent (G. barbadense 3-79). It documented gene expression dynamics by sequencing 1,284 transcriptomes of developing fibers and characterized genetic regulatory perturbations mediated by genomic introgression using a multi-locus model. Introgression of individual homoeologous genes exhibiting extreme low or high expression bias can lead to a parallel expression bias in their non-introgressed duplicates, implying a shared yet divergent regulatory fate of duplicated genes following allopolyploidy. Additionally, the IL N182 with improved fiber quality is characterized, and the candidate gene GhFLAP1 related to fiber length is validated. This study outlines a framework for understanding introgression-mediated regulatory perturbations in polyploids, and provides insights for targeted breeding of superior upland cotton fiber.

Presynaptic quantal size enhancement counteracts post-tetanic release depression.

Repetitive synaptic stimulation can induce different forms of synaptic plasticity but may also limit the robustness of synaptic transmission by exhausting key resources. Little is known about how synaptic transmission is stabilized after high-frequency stimulation.In the present study, we observed that tetanic stimulation of the Drosophila neuromuscular junction (NMJ) decreases quantal content, release-ready vesicle pool size and synaptic vesicle density for minutes after stimulation. This was accompanied by a pronounced increase in quantal size. Interestingly, action potential-evoked synaptic transmission remained largely unchanged. EPSC amplitude fluctuation analysis confirmed the post-tetanic increase in quantal size and the decrease in quantal content, suggesting that the quantal size increase counteracts release depression to maintain evoked transmission. The magnitude of the post-tetanic quantal size increase and release depression correlated with stimulation frequency and duration, indicating activity-dependent stabilization of synaptic transmission. The post-tetanic quantal size increase persisted after genetic ablation of the glutamate receptor subunits GluRIIA or GluRIIB, and glutamate receptor calcium permeability, as well as blockade of postsynaptic calcium channels. By contrast, it was strongly attenuated by pharmacological or presynaptic genetic perturbation of the GTPase dynamin. Similar observations were made after inhibition of the H+-ATPase, suggesting that the quantal size increase is presynaptically driven. Additionally, dynamin and H+-ATPase perturbation resulted in a post-tetanic decrease in evoked amplitudes. Finally, we observed an increase in synaptic vesicle diameter after tetanic stimulation. Thus, a presynaptically-driven quantal size increase, likely mediated by larger synaptic vesicles, counterbalances post-tetanic release depression, thereby conferring robustness to synaptic transmission on the minute time scale. KEY POINTS: Many synapses transmit robustly aftersustained activity despite the limitation of key resources, such as release-ready synaptic vesicles. We report robust synaptic transmission after sustained high-frequency stimulation of the Drosophila neuromuscular junction despite a reduction in release-ready vesicle number. An increased postsynaptic response to individual vesicles, likely driven by an increase in vesicle size due to endocytosis defects, stabilizes synaptic efficacy for minutes after sustained activity. Our study provides novel insights into the mechanisms governing synaptic stability after sustained neural activity.

Activity-dependent transcriptional programs in memory regulate motor recovery after stroke

Stroke causes death of brain tissue leading to long-term deficits. Behavioral evidence from neurorehabilitative therapies suggest learning-induced neuroplasticity can lead to beneficial outcomes. However, molecular and cellular mechanisms that link learning and stroke recovery are unknown. We show that in a mouse model of stroke, which exhibits enhanced recovery of function due to genetic perturbations of learning and memory genes, animals display activity-dependent transcriptional programs that are normally active during formation or storage of new memories. The expression of neuronal activity-dependent genes are predictive of recovery and occupy a molecular latent space unique to motor recovery. With motor recovery, networks of activity-dependent genes are co-expressed with their transcription factor targets forming gene regulatory networks that support activity-dependent transcription, that are normally diminished after stroke. Neuronal activity-dependent changes at the circuit level are influenced by interactions with microglia. At the molecular level, we show that enrichment of activity-dependent programs in neurons lead to transcriptional changes in microglia where they differentially interact to support intercellular signaling pathways for axon guidance, growth and synaptogenesis. Together, these studies identify activity-dependent transcriptional programs as a fundamental mechanism for neural repair post-stroke.

Mapping spatial organization and genetic cell-state regulators to target immune evasion in ovarian cancer

The drivers of immune evasion are not entirely clear, limiting the success of cancer immunotherapies. Here we applied single-cell spatial and perturbational transcriptomics to delineate immune evasion in high-grade serous tubo-ovarian cancer. To this end, we first mapped the spatial organization of high-grade serous tubo-ovarian cancer by profiling more than 2.5 million cells in situ in 130 tumors from 94 patients. This revealed a malignant cell state that reflects tumor genetics and is predictive of T cell and natural killer cell infiltration levels and response to immune checkpoint blockade. We then performed Perturb-seq screens and identified genetic perturbations—including knockout of PTPN1 and ACTR8—that trigger this malignant cell state. Finally, we show that these perturbations, as well as a PTPN1/PTPN2 inhibitor, sensitize ovarian cancer cells to T cell and natural killer cell cytotoxicity, as predicted. This study thus identifies ways to study and target immune evasion by linking genetic variation, cell-state regulators and spatial biology.

Deep learning reveals a damage signalling hierarchy that coordinates different cell behaviours driving wound re-epithelialisation.

One of the key tissue movements driving closure of a wound is re-epithelialisation. Earlier wound healing studies describe the dynamic cell behaviours that contribute to wound re-epithelialisation, including cell division, cell shape changes and cell migration, as well as the signals that might regulate these cell behaviours. Here, we have used a series of deep learning tools to quantify the contributions of each of these cell behaviours from movies of repairing wounds in the Drosophila pupal wing epithelium. We test how each is altered after knockdown of the conserved wound repair signals Ca2+ and JNK, as well as after ablation of macrophages that supply growth factor signals believed to orchestrate aspects of the repair process. Our genetic perturbation experiments provide quantifiable insights regarding how these wound signals impact cell behaviours. We find that Ca2+ signalling is a master regulator required for all contributing cell behaviours; JNK signalling primarily drives cell shape changes and divisions, whereas signals from macrophages largely regulate cell migration and proliferation. Our studies show deep learning to be a valuable tool for unravelling complex signalling hierarchies underlying tissue repair.

Development of a Signal-integrating Reporter to Monitor Mitochondria-ER Contacts.

Mitochondria-endoplasmic reticulum contact sites (MERCS) serve as hotspots for important cellular processes, including calcium homeostasis, phospholipid homeostasis, mitochondria dynamics, and mitochondrial quality control. MERCS reporters based on complementation of green fluorescent proteins (GFP) fragments have been designed to visualize MERCS in real-time, but we find that they do not accurately respond to changes in MERCS content. Here, we utilize split LacZ complementing fragments to develop the first MERCS reporter system (termed SpLacZ-MERCS) that continuously integrates the MERCS information within a cell and generates a fluorescent output. Our system exhibits good organelle targeting, no artifactual tethering, and effective, dynamic tracking of the MERCS level in single cells. The SpLacZ-MERCS reporter was validated by drug treatments and genetic perturbations known to affect mitochondria-ER contacts. The signal-integrating nature of SpLacZ-MERCS may enable systematic identification of genes and drugs that regulate mitochondria-ER interactions. Our successful application of the split LacZ complementation strategy to study MERCS may be extended to study other forms of interorganellar crosstalk.

Retinoic acid induces human gastruloids with posterior embryo-like structures

Gastruloids are a powerful in vitro model of early human development. However, although elongated and composed of all three germ layers, human gastruloids do not morphologically resemble post-implantation human embryos. Here we show that an early pulse of retinoic acid (RA), together with later Matrigel, robustly induces human gastruloids with posterior embryo-like morphological structures, including a neural tube flanked by segmented somites and diverse cell types, including neural crest, neural progenitors, renal progenitors and myocytes. Through in silico staging based on single-cell RNA sequencing, we find that human RA-gastruloids progress further than other human or mouse embryo models, aligning to E9.5 mouse and CS11 cynomolgus monkey embryos. We leverage chemical and genetic perturbations of RA-gastruloids to confirm that WNT and BMP signalling regulate somite formation and neural tube length in the human context, while transcription factors TBX6 and PAX3 underpin presomitic mesoderm and neural crest, respectively. Looking forward, RA-gastruloids are a robust, scalable model for decoding early human embryogenesis.

The genetic landscape of antibiotic sensitivity in Staphylococcus aureus.

Despite the critical importance of essential genes, systems-level investigations of their contribution to antibiotic sensitivity have been limited. Using CRISPR Adaptation-mediated Library Manufacturing (CALM), we generated ultra-dense CRISPR interference (CRISPRi) libraries in methicillin-sensitive and -resistant strains of Staphylococcus aureus, which allowed us to quantify gene fitness on a global scale across ten clinically relevant antibiotics. This led to the identification of a comprehensive set of known and novel biological processes modulating bacterial fitness in the antibiotics. Notably, we found that essential genes from diverse processes dominated antibiotic-gene interactions, including a large number of synergistic interactions between bactericidal antibiotics and processes such as cell wall synthesis/cell division (CC), DNA replication/DNA recombination (DD), protein export, and coenzyme A biosynthesis. Simultaneous genetic perturbations of diverse CC and DD processes aggravated bacterial fitness, revealing a widespread synergy between the two highly coordinated processes. In contrast, perturbation of transcriptional, translational, and select energy processes antagonized the effects of bactericidal antibiotics. Finally, we show that small molecule inhibitors recapitulated synergistic antibiotic-gene interactions, providing a rational foundation for developing novel combinatorial antimicrobial therapies.

Uncovering underlying physical principles and driving forces of cell differentiation and reprogramming from single-cell transcriptomics

Recent advances in single-cell sequencing technology have revolutionized our ability to acquire whole transcriptome data. However, uncovering the underlying transcriptional drivers and nonequilibrium driving forces of cell function directly from these data remains challenging. We address this by learning cell state vector fields from discrete single-cell RNA velocity to quantify the single-cell global nonequilibrium driving forces as landscape and flux. From single-cell data, we quantified the Waddington landscape, showing that optimal paths for differentiation and reprogramming deviate from the naively expected landscape gradient paths and may not pass through landscape saddles at finite fluctuations, challenging conventional transition state estimation of kinetic rate for cell fate decisions due to the presence of the flux. A key insight from our study is that stem/progenitor cells necessitate greater energy dissipation for rapid cell cycles and self-renewal, maintaining pluripotency. We predict optimal developmental pathways and elucidate the nucleation mechanism of cell fate decisions, with transition states as nucleation sites and pioneer genes as nucleation seeds. The concept of loop flux quantifies the contributions of each cycle flux to cell state transitions, facilitating the understanding of cell dynamics and thermodynamic cost, and providing insights into optimizing biological functions. We also infer cell-cell interactions and cell-type-specific gene regulatory networks, encompassing feedback mechanisms and interaction intensities, predicting genetic perturbation effects on cell fate decisions from single-cell omics data. Essentially, our methodology validates the landscape and flux theory, along with its associated quantifications, offering a framework for exploring the physical principles underlying cellular differentiation and reprogramming and broader biological processes through high-throughput single-cell sequencing experiments.