Biology subjects

Hsu, C.-W.

Publications and source records attributed to Hsu, C.-W..

15 recordsLinked to original sources

bioRxiv2025

Host-derived lipid transfer and metabolic reprogramming in a ciliate-algal symbiosis

Symbiotic associations enable species to integrate complementary traits and adapt to novel environments. However, how the host and endosymbiont exchange nutrients remains poorly understood in most cases. Previously Paramecium bursaria cells were shown to reorganize lipid droplets to accommodate endosymbiotic Chlorella cells and interfering with lipid metabolism reduced the endosymbiont number. Here, we combined transcriptomics, lipidomics, imaging mass spectrometry, and stable-isotope tracing to investigate organic nutrient exchange in this symbiotic system. Our results reveal that endosymbiotic algae undergo extensive reprogramming of lipid metabolic pathways and accumulate markedly higher levels of triglycerides than free-living algae. Isotope-labeling experiments demonstrate that at least some of these lipids originate from the host, providing direct evidence for organic carbon transfer from Paramecium to its algal endosymbionts. Together, our results show that the establishment of symbiosis fundamentally reshapes algal lipid metabolism and uncover an unexpected host-to-symbiont lipid provisioning mechanism--opposite to the canonical direction of carbon flow observed in most photosynthetic symbioses. This work provides new insight into the metabolic principles that sustain and stabilize endosymbiotic partnerships.

microbiology↗

bioRxiv2025

GeneSys: Generative Modeling of Developmental System

Temporal single-cell transcriptomics enables the reconstruction of dynamic gene expression changes during development. Yet, its analytical power is often limited by data sparsity, technical noise, and imbalanced representation of cell types across time points. To overcome these challenges, we present GeneSys (Generative Modeling of Developmental System), a generative deep learning model that simulates single-cell transcriptomic landscapes under developmental constraints, which is informed by prior biological knowledge or user-defined hypotheses. GeneSys integrates a temporal variational autoencoder with a cell-type classifier, requiring a lineage blueprint as input, which enables it to model the temporal transitions of transcriptional states with cell-type specificity. Leveraging data from Arabidopsis thaliana roots and mouse embryos, we show that GeneSys learns robust developmental trajectories, generates imputed and representative transcriptomes, and enhances gene prioritization accuracy compared to unregularized scRNA-seq data. By applying gene masking and augmentation, GeneSys reveals interpretable gene expression programs (GEPs) and serves as an in silico platform to test the impact of specific genes or gene sets on user-defined developmental outcomes. Additionally, GeneSys computes linear interaction matrices (LIMAs) to infer dynamic gene networks and prioritize transcription factors with spatiotemporal resolution. These features enable GeneSys to nominate key genes governing state transitions in a developmental system, supporting both mechanistic insight and hypothesis generation. Together, GeneSys provides a flexible and extensible framework to denoise single-cell data and simulate transcriptomic developmental landscape guided by known or hypothesized developmental constraints, empowering the discovery of regulatory mechanisms from high-dimensional single-cell datasets.

systems biology↗

bioRxiv2025

Optimization of Tissue Clearing Methods and Imaging Conditions for 3D Visualization of the Vasculature of the Adult Murine Knee

The vasculature is an essential regulator of tissue oxygenation, metabolism, and immune surveillance under developmental and homeostatic conditions, as well as in regeneration and disease. Within the musculoskeletal system, blood vessels are involved in bone development and resorption, and they also mediate the inflammatory processes that contribute to diseases affecting the joints, including osteoarthritis. Historically, visualization of vascular networks in joints has been limited to conventional histological approaches due to technical limitations and the inherit structure of the skeletal muscle and bones. Recent advances in optical tissue clearing technologies and 3D fluorescence imaging approaches enabling three-dimensional analysis in whole, intact tissues offer an entree to interrogate musculoskeletal development and disease at a cellular resolution. However, these techniques were not originally developed to image hard mineralized tissues, such as the femur and tibias. To circumvent these challenges, we have optimized tissue decalcification conditions and systematically compared aqueous- and solvent-based tissue clearing approaches to establish an optimal pipeline for clearing and imaging of mineralized tissues with superior resolution of the vasculature. Collectively, this work shows that optical clearing and lightsheet microscopy presents a viable method for generating high-resolution images of the murine hindlimb vasculature, with potential applications in aging and disease modeling.

developmental biology↗

bioRxiv2025

Learning and language in the unconscious human hippocampus

Consciousness is a fundamental component of cognition,1 but the degree to which higher-order pattern recognition relies on it remains disputed.2,3 Here we demonstrate the persistence of oddball discrimination, semantic processing, and online prediction in individuals under general anesthesia-induced loss of consciousness.4,5 Using high-density Neuropixels microelectrodes6 to record both single unit and local field potential neural activity in the human hippocampus while playing a series of tones to anesthetized patients, we found that hippocampal neurons and local oscillations retained some detection of oddball tones. This effect size grew over the course of the experiment ([~]10 minutes), demonstrating representational plasticity. A biologically plausible recurrent neural network model showed that learning and oddball representation are an emergent property of flexible tone discrimination. Moreover, when we played language stimuli, single units and local field potentials carried information about the semantic and grammatical features of natural speech, even predicting semantic information about upcoming words. Together these results indicate that in the hippocampus, which is anatomically and functionally distant from primary sensory cortices,7 complex processing of sensory stimuli occurs even in the unconscious state.

neuroscience↗

bioRxiv2024

Sensitive dissection of a genomic regulatory landscape using bulk and targeted single-cell activation

Transcriptional enhancers are non-coding DNA elements that regulate gene transcription in a temporal and tissue-specific manner. Despite advances in computational and experimental methods, identifying enhancers and their target genes essential for specific biological processes remains challenging. Determining target genes for enhancers is also complex and often relies on indirect, low-resolution, and/or assumptive methodologies. To identify and functionally perturb enhancers at their endogenous sites without altering their sequence, we performed a pooled tiling CRISPR activation (CRISPRa) screen surrounding PHOX2B, a master regulator of neuronal cell fate and a key player in neuroblastoma development. This screen allowed the de novo identification of CRISPRa responsive elements (CaREs) that alter cellular growth within the 2 Mb genomic region. To determine CaRE target genes, we developed TESLA-seq (TargEted SingLe cell Activation), which combines CRISPRa screening with targeted single-cell RNA-sequencing and enables the parallel readout of the effect of hundreds of enhancers on all genes in the locus. While most TESLA-revealed CaRE-gene relationships involved neuroblastoma-related regulatory elements already active in the system, we found many CaREs and target connections normally active only in other tissue types or with no previous evidence and induced out of context by CRISPRa. This highlights the power of TESLA-seq to reveal gene regulatory networks, including edges active outside of a given experimental system. HighlightsO_LISystematically perturbed regulatory landscape in a 2 Mb genomic region surrounding PHOX2B to identify hundreds of CRISPRa-responsive elements that affect cellular growth C_LIO_LIDeveloped TESLA-seq as a principled molecular approach to find gene targets of dozens of candidate regulatory elements C_LIO_LIValidated interactions between identified regulatory elements and target genes and characterized their genomic features C_LIO_LIIntegrated a compendium of epigenomic datasets to identify regulatory relationships induced out of context or with no previous evidence C_LI

genomics↗

bioRxiv2024

A Comprehensive Atlas of AAV Tropism in the Mouse

Gene therapy with Adeno-Associated Viral (AAV) vectors requires knowledge of their tropism within the body. Here we analyze the tropism of ten naturally occurring AAV serotypes (AAV3B, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAVrh8, AAVrh10 and AAVrh74) following systemic delivery into male and female mice. A transgene expressing ZsGreen and Cre recombinase was used to identify transduction in a cell-dependent manner based on fluorescence. Cre-driven activation of tdTomato fluorescence offered superior sensitivity for transduced cells. All serotypes except AAV3B and AAV4 had high liver tropism. Fluorescence activation revealed transduction of unexpected tissues, including adrenals, testes and ovaries. Rare transduced cells within tissues were also readily visualized. Biodistribution of AAV genomes correlated with fluorescence, except in immune tissues. AAV4 was found to have a pan-endothelial tropism while also targeting pancreatic beta cells. This public resource enables selection of the best AAV serotypes for basic science and preclinical applications in mice.

molecular biology↗

bioRxiv2024

The brassinosteroid receptor gene BRI1 safeguards cell-autonomous brassinosteroid signaling across tissues

Brassinosteroid signaling is essential for plant growth as exemplified by the dwarf phenotype of loss-of-function mutants in BRASSINOSTEROID INSENSITIVE 1 (BRI1), a ubiquitously expressed Arabidopsis brassinosteroid receptor gene. Complementation of brassinosteroid-blind receptor mutants by BRI1 expression with various tissue-specific promoters implied that local brassinosteroid signaling may instruct growth non-cell-autonomously. Here we performed such rescues with a panel of receptor variants and promoters, in combination with tissue-specific transgene knockouts. Our experiments demonstrate that brassinosteroid receptor expression in several tissues is necessary but not sufficient for rescue. Moreover, complementation with tissue-specific promoters requires the genuine BRI1 gene body sequence, which confers ubiquitous expression of trace receptor amounts that are sufficient to promote brassinosteroid-dependent root growth. Our data, therefore, argue for a largely cell-autonomous action of brassinosteroid receptors.

plant biology↗

bioRxiv2022

Brassinosteroid gene regulatory networks at cellular resolution

Brassinosteroids (BRs) are plant steroid hormones that regulate diverse processes such as cell division and cell elongation. BRs control thousands of genes through gene regulatory networks that vary in space and time. By using time-series single-cell RNA-sequencing to identify BR-responsive gene expression specific to different cell types and developmental stages of the Arabidopsis root, we uncovered the elongating cortex as a site where BRs trigger a shift from proliferation to elongation associated with increased expression of cell wall-related genes. Our analysis revealed HAT7 and GTL1 as BR-responsive transcription factors that regulate cortex cell elongation. These results establish the cortex as an important site for BR-mediated growth and unveil a BR signaling network regulating the transition from proliferation to elongation, illuminating new aspects of spatiotemporal hormone response.

plant biology↗

bioRxiv2022

Single-cell transcriptomics of the Arabidopsis floral abscission zone

Abscission is the programmed separation of plant organs. It is widespread in the plant kingdom with important functions in development and environmental response. In Arabidopsis, abscission of floral organs (sepals, petals, and stamens) is controlled by two receptor-like protein kinases HAESA (HAE) and HAESA LIKE-2 (HSL2), which orchestrate the programmed dissolution of the abscission zone connecting floral organs to the developing fruit. In this work, we use single-cell RNA-Sequencing to characterize the core HAE/HSL2 abscission gene expression program. We identify the MAP KINASE PHOSPHATASE-1/MKP1 gene as a negative regulator of this pathway. MKP1 acts prior to activation of HAE/HSL2 signaling to establish a signaling threshold required for the initiation of abscission. By analogy to electrical circuit control, we liken MKP1 to a molecular grounding mechanism that dissipates errant pathway activation absent HAE/HSL2 signaling. Furthermore, we use single-cell data to identify genes expressed in two sub-populations of abscission zone cells: those proximal and those distal to the plane of separation. We identify INFLORESCENCE DEFICIENT IN ABSCISSION/IDA, encoding the activating ligand of HAE/HSL2, as one of the mRNAs most highly enriched in distal abscission zone cells at the base of the abscising organs. We show how this expression pattern forms a biophysical circuit breaker whereby, when the organ is shed, the source of the IDA peptide is removed, leading to cessation of HAE/HSL2 signaling. Overall, this work provides insight into the multiple control mechanisms acting on the abscission-signaling pathway.

plant biology↗

bioRxiv2022

Live cell biosensors based on the fluorescence lifetime of environment-sensing dyes

Most biosensors used with fluorescence lifetime imaging (FLIM) have been based on fluorescence resonance energy transfer (FRET). Here we examined the capabilities of FLIM biosensors based on environment-sensing dyes. We screened merocyanine dyes to find an optimal combination of environment-induced FLIM changes, photostability and brightness at wavelengths suitable for live cell imaging. A biosensor reporting conformational changes of endogenous Cdc42 protein was tested in vitro and in live cells. In addition to the known advantages of FLIM (quantitation independent of photobleaching, intracellular biosensor distribution, and excitation intensity), the spectral properties of the merocyanine-FLIM biosensor (mcFLIM) provided enhanced sensitivity at low activation levels (<10%) where FRET is typically least sensitive. We leveraged these properties and the phasor representation of FLIM to determine the specific concentration of activated Cdc42 across the cell.

cell biology↗

bioRxiv2022

EZ Clear for simple, rapid, and robust mouse whole organ clearing

Tissue clearing for whole organ cell profiling has revolutionized biology and imaging for exploration of organs in three-dimensional space without compromising tissue architecture. But complicated, laborious procedures, or expensive equipment, as well as the use of hazardous, organic solvents prevents the widespread adoption of these methods. Here we report a simple and rapid tissue clearing method, EZ Clear, that can clear whole adult mouse organs in 48 hours in just three simple steps. Samples stay at room temperature and remain hydrated throughout the clearing process, preserving endogenous and synthetic fluorescence, without altering sample size. After wholemount clearing and imaging, EZ Cleared samples can be further processed for downstream embedding and cryosectioning followed by standard histology or immunostaining, without loss of endogenous or synthetic fluorescence signal. Overall, the simplicity, speed, and flexibility of EZ Clear make it easy to adopt and apply to diverse approaches in biomedical research.

developmental biology↗

bioRxiv2020

A single cell Arabidopsis root atlas reveals developmental trajectories in wild type and cell identity mutants

SummaryCell fate acquisition is a fundamental developmental process in all multicellular organisms. Yet, much is unknown regarding how a cell traverses the pathway from stem cell to terminal differentiation. Advances in single cell genomics1 hold promise for unraveling developmental mechanisms2–3 in tissues4, organs5–6, and organisms7–8. However, lineage tracing can be challenging for some tissues9 and integration of high-quality datasets is often necessary to detect rare cell populations and developmental states10,11. Here, we harmonized single cell mRNA sequencing data from over 110,000 cells to construct a comprehensive atlas for a stereotypically developing organ with indeterminate growth, the Arabidopsis root. To test the utility of the atlas to interpret new datasets, we profiled mutants for two key transcriptional regulators at single cell resolution, shortroot and scarecrow. Although both transcription factors are required for early specification of cell identity12, our results suggest the existence of an alternative pathway acting in mature cells to specify endodermal identity, for which SHORTROOT is required. Uncovering the architecture of this pathway will provide insight into specification and stabilization of the endodermis, a tissue analogous to the mammalian epithelium. Thus, the atlas is a pivotal advance for unraveling the transcriptional programs that specify and maintain cell identity to regulate organ development in space and time.Competing Interest StatementPNB is the co-founder and Chair of the Scientific Advisory Board of Hi Fidelity Genetics, Inc, a company that works on crop root growth.View Full Text

plant biology↗

bioRxiv2019

Human and mouse essentiality screens as a resource for disease gene discovery

Although genomic sequencing has been transformative in the study of rare genetic diseases, identifying causal variants remains a considerable challenge that can be addressed in part by new gene-specific knowledge. Here, we integrate measures of how essential a gene is to supporting life, as inferred from the comprehensive viability and phenotyping screens performed on knockout mice by the International Mouse Phenotyping Consortium and from human cell line essentiality screens. We propose a novel, cross-species gene classification across the Full Spectrum of Intolerance to Loss-of-function (FUSIL) and demonstrate that genes in five mutually exclusive FUSIL categories have differing characteristics in the biological processes they regulate, tissue expression levels and human mutation rates. Most notably, Mendelian disease genes, particularly those associated with developmental disorders, are highly overrepresented in the developmental lethal category, representing genes not essential for cell survival but required for organism development. Exploiting this finding, we have screened developmental disorder cases from three independent disease sequencing consortia and identified potentially pathogenic, de novo variants shared in different patients for several developmental lethal genes that have not previously been associated with rare disease. We therefore propose FUSIL as an efficient resource for disease gene discovery.

genomics↗

bioRxiv2019

Soft Windowing Application to Improve Analysis of High-throughput Phenotyping Data

MotivationHigh-throughput phenomic projects generate complex data from small treatment and large control groups that increase the power of the analyses but introduce variation over time. A method is needed to utlize a set of temporally local controls that maximises analytic power while minimising noise from unspecified environmental factors.\n\nResultsHere we introduce \"soft windowing\", a methodological approach that selects a window of time that includes the most appropriate controls for analysis. Using phenotype data from the International Mouse Phenotyping Consortium (IMPC), adaptive windows were applied such that control data collected proximally to mutants were assigned the maximal weight, while data collected earlier or later had less weight. We applied this method to IMPC data and compared the results with those obtained from a standard non-windowed approach. Validation was performed using a resampling approach in which we demonstrate a 10% reduction of false positives from 2.5 million analyses. We applied the method to our production analysis pipeline that establishes genotype-phenotype associations by comparing mutant versus control data. We report an increase of 30% in significant p-values, as well as linkage to 106 versus 99 disease models via phenotype overlap with the soft windowed and non-windowed approaches, respectively, from a set of 2,082 mutant mouse lines. Our method is generalisable and can benefit large-scale human phenomic projects such as the UK Biobank and the All of Us resources.\n\nAvailability and ImplementationThe method is freely available in the R package SmoothWin, available on CRAN http://CRAN.R-project.org/package=SmoothWin.

bioinformatics↗

bioRxiv2018

On the post-glacial spread of human commensal Arabidopsis thaliana: journey to the east

With the availability of more sequenced genomes, our understanding of the evolution and demographic history of the model plant Arabidopsis thaliana is rapidly expanding. Here we compile previously published data to investigate global patterns of genetic variation. While the Southeast African accessions were reported to be the most divergent among worldwide populations, we found accessions from Yunnan, China to be genetically close to the sub-Saharan accessions. Our further investigation of worldwide chloroplast genomes identified several deeply diverged haplogroups existing only in Eurasia, and the African populations have lower variation in many haplogroups they shared with the Eurasian populations. Bayesian inferences of chloroplast demography showed that representative haplogroups of Africa exhibited long-term stable population size, suggesting recent selective sweep or bottleneck is not able to explain the lower chloroplast variation in Africa. Taken together, these patterns cannot be easily explained by a single out-of-Africa event. Several Eurasian chloroplast haplogroups had rapid population growth since 10 kya, presumably reflecting the recent expansion of the weedy non-relicts across Eurasia. Our demographic analysis on a chromosomal region un-affected by relict introgression also suggested the European, Central Asian, and Chinese Yangtze populations diverged no earlier than 15 kya, in contrast to previous estimates of 45 kya inferred from whole genome that likely contains relict admixture. The most recent expansion is observed in the Yangtze population of China less than 2000 years ago. Similar to Iberia, the western end of non-relict expansion reported in our previous study, in this eastern end of Eurasia we find clear traces of gene flow between the Yangtze non-relicts and the Yunnan relicts. Genes under strong selection and previously suggested to contribute to adaptation in the Yangtze valley are enriched for traces of relict introgression, especially those related with biotic and immune responses. The results suggest the ability of non-relicts to obtain locally adaptive alleles through admixture with relicts is an important factor contributing to the rapid expansion across the environmental gradients spanning the eastern to the western coast of Eurasia.

evolutionary biology↗