Biology subjects

Li, C.

Publications and source records attributed to Li, C..

At least 73 records · Page 4Linked to original sources

bioRxiv2026

Evolutionary Origins and Synthetic Reconstruction of Gingerols

How new enzymatic activities become integrated with existing metabolic networks to generate lineage-restricted plant chemistry remains a central question in metabolic evolution, exemplified by gingerol biosynthesis in Zingiberaceae, whose complete enzymatic basis has remained elusive. Here, we resolve this problem by elucidating the evolutionary origins and the full biosynthetic pathway of gingerol through single-cell transcriptomics, functional screening, structural biology, comparative genomics and pathway reconstruction. A coordinated metabolic program operates in ginger oil cells, thereby guiding the identification of diketide-CoA synthases, gingerol synthases (GS) and gingerol reductases that together constitute the complete pathway. GS remodels the conserved type III polyketide synthase scaffold to organize a fatty acyl-CoA and a phenylpropanoid-derived intermediate for gingerol formation. Furthermore, GS arose through duplication and limited active-site remodeling of a curcumin synthase-like enzyme, accompanied by coordinated recruitment of precursor-supply and reduction pathways. Translating these principles into yeast achieves the first de novo microbial production of 6-gingerol from glucose at 12.4 mg/L. These findings reveal the evolutionary logic by which plants recruit and coordinate novel enzymatic activities to establish lineage-restricted metabolic program, providing a framework for understanding and engineering specialized metabolism across the plant kingdom.

synthetic biology↗

bioRxiv2026

Photosynthetic assimilate determines branch size and biomass more than branch number in Arabidopsis

Shoot branching is a primary determinant of plant form and crop yield, yet whether auxin or carbon supply is the proximal regulator of branching remains contested. In Arabidopsis, an earlier report that exogenous auxin fails to restore apical dominance after decapitation has been taken to weaken the case for auxin, and several studies have proposed that sugars are the primary regulator. Resolving this has been difficult because most perturbations of sugar status also disturb auxin. Here, we revisit the control of Arabidopsis branching using well-controlled, largely unperturbed plants and approaches designed to isolate each pathway. Contrary to the earlier report, apically applied auxin restored the suppression of rosette branching after decapitation, placing Arabidopsis in line with other species. In a dataset of 718 plants, cauline and rosette branching were weakly but significantly negatively correlated, consistent with a polar-auxin-transport-based model and contrary to a previous conclusion of no relationship. Removing all rosette leaves at bolting slowed bud growth but did not alter the final number of branches. Varying photosynthetic photon flux density across six natural accessions, analyzed by piecewise structural equation modeling, showed that photoassimilate acted far more strongly on the mass deposited into branches than on whether a bud initiates a branch. We conclude that auxin remains a major regulator of apical dominance in Arabidopsis, and that photosynthetic assimilate, while required as a substrate for branch growth, contributes little to determining branch number but more to branch size and biomass.

plant biology↗

bioRxiv2026

First Order Associations Between Banff Acute Lesions in Kidney Allograft Biopsies and a Urinary Cell Three-Gene Diagnostic Signature

Banff acute lesion scores underpin histologic classification of kidney allograft biopsies; however, biomarker studies rely on second-order associations with diagnostic categories that introduce confounding. We quantified the first-order relationships between Banff acute lesion scores and the validated urinary cell three-gene rejection signature. In 354 biopsy-urine pairs, three-gene signature scores computed using a locked regression equation incorporating absolute copy numbers of CD3E mRNA, CXCL10 mRNA, and 18S rRNA in urinary cell RNA-were related to glomerulitis (g), peritubular capillaritis (ptc), interstitial inflammation (i), and tubulitis (t). Signature scores rose monotonically with Banff acute lesion severity, with 1.5 to 1.8-fold higher odds of more severed g, ptc, i, and t (all P<0.0001), and showed good calibration. Associations remained robust for composite microvascular (g+ptc) and tubulointerstitial (i+t) indices and were strongest for severe g and t, supporting this signature as a noninvasive, quantitative readout of acute rejection pathology with immediate diagnostic applicability.

pathology↗

bioRxiv2026

Population structure, genetic diversity and global migration of the banana Fusarium wilt pathogen, Fusarium oxysporum f. sp. cubense tropical race 4

O_LIThe current epidemic of banana Fusarium wilt is caused by Fusarium oxysporum f. sp. cubense tropical race 4 (Foc TR4). Foc TR4 originates in Asia and was first reported in the early 1990s, whereafter it was restricted to five Asian countries and the Northern Territory of Australia for two decades. Since 2013, the fungus was detected in the Middle East, the Indian subcontinent, Africa and Latin America. Its recent spread may be caused by the expansion of Cavendish banana production and the movement of contaminated planting materials. C_LIO_LIWhole genome sequences of 119 Foc TR4 isolates were compared based on single nucleotide polymorphisms (SNPs) to assess the population structure, genetic diversity and relation among isolates from 20 countries to reveal the evolutionary origin and likely migration. C_LIO_LIFoc TR4 is a monophyletic lineage divided into four clusters. Two of the clusters included isolates mostly from eastern Indonesia and seem to be ancestral with many rare alleles and high genetic diversity supporting this region as centre of origin. The remaining clusters were less diverse and associated with pandemic expansion. C_LIO_LIKnowledge on Foc TR4 emergence and dispersal can be used to inform more effective biosecurity and management practice. C_LI

evolutionary biology↗

bioRxiv2026

Who Funds Open Data Sharing? Analysis of data availability statements in biomedical publications

Open data sharing is increasingly mandated by research funders, journals, and institutions, yet large-scale compliance measurement remains challenging. We analyzed 951,949 open access biomedical research articles published between January 2024 and June 2025 using a dual-source pipeline: PDF-based text extraction (MinerU) where PDFs were available and PMC XML otherwise, followed by algorithmic detection of data sharing statements (oddpub v7.2.3), enriched with funder, journal, and institutional metadata from OpenAlex. The corpus included 294,172 PDF-covered articles (30.9%) and 657,777 XML-only articles (69.1%). We found an over-all open data rate of 8.7%, rising to 11.7% among funder-linked articles (those with at least one funder identified in the metadata). Rates varied more than tenfold across the research ecosystem: leading major funders reached observed open data rates of 20-24%, while top journals reached observed rates of 70-86%, with corrected estimates as high as 92.9% (Nature Genetics) after adjusting for XML-only coverage limitations. PDF-based detection identified approximately 52% more data sharing statements than XML-based methods on the same articles. These observed rates differ markedly across funders and journals, and current overall sharing remains far below universal compliance. These patterns provide an empirical baseline against which future policy changes can be measured. An interactive dashboard at https://www.opensciencemetrics.org enables stakeholders to explore and benchmark these results.

scientific communication and education↗

bioRxiv2026

Chemokine-Dependent Natural Killer Cells Prevent Pulmonary Metastasis in a Newly Established Mouse Osteosarcoma Model

Rampant genomic instability of osteosarcoma (OS) and associated inter- and intratumoral heterogeneity convey a high risk of metastasis despite contemporary multimodal therapy. A mouse primary OS tumor model, originating from Myc-overexpressing Trp53-null mesenchymal progenitor cells, closely mimics cardinal genetic features and gene expression patterns of human OS samples. Subcutaneous injection of OS cells into immunocompromised NSG mice produced extensive metastases, whereas many fewer metastases occurred in T cell-deficient nude mice, suggesting a principal role for innate immunity in controlling OS dissemination. Depletion of natural killer (NK) cells in nude mice facilitated OS metastasis. OS cells released a suite of chemokines, with CCL2 the most prominent. A genome-wide CRISPR/Cas9 screen in OS cells identified 11 genes, including Ccl2, whose loss facilitated pulmonary metastasis in nude mice. Disruption of CCL2 in OS cells partially phenocopied the effects of antibody-dependent NK cell depletion, underscoring a plausible OS-NK signaling pathway that limits OS metastasis. SignificanceOsteosarcoma exhibits complex genomic instability and a propensity for pulmonary metastasis that limit chemotherapeutic response and patient survival. Despite the plethora of heterogeneous genetic alterations that connote poor prognosis, a novel preclinical in vivo model for studying metastasis highlights potentially targetable signaling between osteosarcoma cell-derived chemokines and pulmonary NK cells.

cancer biology↗

bioRxiv2026

AMPKγ2 Deacetylation Drives Nuclear Translocation and Doxorubicin-Induced Cardiomyopathy via Nucleolar Stress Signaling

Doxorubicin (Dox)-induced cardiomyopathy (DIC), characterized by cardiomyocyte apoptosis, remains a major clinical challenge in chemotherapy. The regulatory {gamma}2 subunit of AMP-activated protein kinase (AMPK{gamma}2) plays a key role in cardiovascular diseases, but its function in DIC is poorly understood. Here, we report that Dox induces isoform-specific deacetylation and nuclear accumulation of {gamma}2, triggering nucleolar stress and p53-mediated apoptosis. Mechanistically, HDAC3 and TIP60 interact with {gamma}2 and modulate the acetylation of multiple lysine residues within its nuclear localization signal (NLS), controlling its nucleocytoplasmic shuttling. Dox enhances HDAC3-mediated {gamma}2 deacetylation, thereby driving nuclear accumulation of the {gamma}2-containing AMPK ({gamma}2-AMPK) while suppressing the cytosolic AMPK activity. Nuclear {gamma}2-AMPK phosphorylates and inactivates TIF-IA, a key RNA polymerase I-specific transcription initiation factor, leading to nucleolar stress through inhibition of rRNA transcription. rRNA deficit triggers release of free ribosomal proteins (RPs), which bind to and inhibit the E3 ubiquitin ligase MDM2, resulting in p53 stabilization and activation of apoptotic signaling. Using genetically engineered cardiomyocytes and a DIC mouse model, we found that a deacetyl-mimetic {gamma}2 mutant (6KR) exacerbated DIC, whereas an acetyl-mimetic mutant (6KQ) was cardioprotective. Collectively, our findings establish acetylation-driven nuclear translocation of {gamma}2 as a critical node linking Dox-induced nucleolar stress to p53-dependent apoptosis and suggest a promising cardio-oncology strategy that combines HDAC inhibitors with Dox to mitigate DIC. Significance statementDoxorubicin is an effective cancer drug, but its use is limited by cardiomyopathy. Our study reveals that doxorubicin drives HDAC3-mediated deacetylation of AMPK{gamma}2, exposing its nuclear localization signal and redirecting {gamma}2-containing AMPK from the cytoplasm to the nucleus. Nuclear AMPK{gamma}2 phosphorylates TIF-IA, suppresses ribosomal RNA synthesis, and activates a nucleolar stress pathway that stabilizes p53 and promotes cardiomyocyte apoptosis. In mice, a deacetylation-mimetic AMPK{gamma}2 mutant worsens doxorubicin-induced cardiomyopathy, whereas an acetylation-mimetic mutant is protective. These findings uncover an acetylation-controlled spatial switch in AMPK signaling and identify the AMPK{gamma}2 deacetylation-nucleolar stress axis as a potential target for reducing chemotherapy-associated cardiac injury.

cell biology↗

bioRxiv2026

PFOS aggravates atherosclerosis via Bacteroides caecimuris expansion-driven bile acid remodeling and subsequent intestinal FXR-TLR3 signaling cascade

BACKGROUNDPerfluorooctane sulfonate (PFOS) is a widely distributed persistent organic pollutant in the environment and has been associated with an increased risk of atherosclerosis. However, the underlying pathogenic mechanisms remain largely unclear. This study aimed to investigate the effects of PFOS on atherosclerosis and its associated gut-vascular axis. METHODSPseudo-germ-free mouse models and fecal microbiota transplantation (FMT) were used to determine the role of the gut microbiota in PFOS-induced atherosclerosis. Metagenomic sequencing was performed to characterize alterations in gut microbial composition following PFOS exposure, and targeted metabolomics was used to assess bile acid profiles in the ileum and plasma. Transcriptomic analysis of Bacteroides caecimuris (B.caecimuris) was conducted to explore the reasons for the increased abundance of B.caecimuris after PFOS exposure. In addition, intestinal transcriptomics and ChIP-qPCR were performed to validate transcriptional regulation within the FXR-TLR3 signaling axis. RESULTSAmong 127 participants with paired serum and fecal samples, including 82 patients undergoing coronary angiography with Gensini scores (GS score), fecal PFOS levels were significantly associated with lipid profiles and GS score, whereas serum PFOS showed no such association.Mechanistically, PFOS exposure promotes intestinal enrichment of B.caecimuris by upregulating its tolC gene, thereby enhancing efflux capacity.This microbial shift was accompanied by reduced levels of tauro-ursodeoxycholic acid (TUDCA) and aberrant activation of intestinal FXR signaling.Further analyses demonstrated that FXR activation upregulated TLR3 expression and promoted inflammatory responses and atherosclerosis progression via the TLR3-NF-{kappa}B signaling axis. Both intestinal epithelial-specific FXR deficiency (Fxr{Delta}IE) and TUDCA supplementation significantly suppressed pathway activation and alleviated disease phenotypes.Functional experiments identified TLR3 as a key downstream effector of FXR. Overexpression of TLR3 abolished the protective effects observed in Fxr{Delta}IE mice. Moreover, pharmacological inhibition of TLR3 using CU CPT-4a significantly improved established atherosclerotic lesions in vivo. CONCLUSIONSThis study identifies a gut microbiota-driven FXR-TLR3 signaling axis that mediates PFOS-induced atherosclerosis. These findings provide new mechanistic insights into environmentally induced cardiovascular disease and suggest potential targets for risk assessment and therapeutic intervention.

pathology↗

bioRxiv2026

CD117 epitope-shielded hematopoietic stem cell transplantation with toxin-free conditioning and in vivo selection ameliorates β-thalassemia model

Clinical evidence demonstrates that ex vivo gene therapy and genome engineering of hematopoietic stem and progenitor cells (HSPCs) could represent one-time cures. However, while genome editing itself has become increasingly efficient and precise, the toxic conditioning required for hematopoietic stem cell transplantation remains a major barrier to broad clinical implementation of these otherwise curative therapies. In particular, the use of busulfan for myeloablative conditioning constitutes a major safety concern. While preclinical studies established CD117 as a promising target for antigen-specific therapy, clinical translation faced setbacks balancing efficacy and safety. To overcome current limitations, we generated a new CD117-blocking monoclonal antibody (CIM058) and demonstrate its potency to block wild-type HSPCs. To enable long-term blockade of host HSPCs even after transplantation, we used prime editing to engineer CIM058-resistant human CD34+ HSPCs. When combined, CIM058 and the epitope engineered CD34+ HSPCs ameliorated disease phenotype in a {beta}-thalassemia model. Our results suggest that this approach may overcome the reliance on busulfan or other myeloablative conditioning regimens with their associated morbidities, and by enabling toxin-free conditioning and in vivo selection of edited cells, may facilitate clinical implementation of these highly valuable genetic therapies.

bioengineering↗

bioRxiv2026

Rapid immunostaining and high-resolution three-dimensional light-sheet microscopy of intact calcified tissues

High-resolution 3D imaging is an important strategy for visualizing and analysing complex skeletal tissue architecture and the bone marrow microenvironment. However, multicolor immunolabeling and imaging of intact skeletal tissues are technologically challenging. The current immunolabeling and clearing methods for intact skeletal elements are very limited, time-consuming and generate low-resolution data or depend on the use of reporter mice. Here, we describe a protocol for efficient clearing and immunolabeling of intact calcified tissues that enables superfast, single-cell resolution, and quantitative 3D light-sheet imaging of intact skeletal elements and teeth. A key aspect of our protocol is the addition of a collagenase digestion step after fixation and decalcification. This step enhances antibody penetration, resulting in deep, comprehensive staining throughout immunostained bones and other calcified tissues. The protocol includes soft tissue removal, fixation, decalcification, bone dehydration, and bleaching, followed by antigen retrieval and permeabilization before the collagenase digestion step. This procedure is performed to prepare the samples for the tissue clearing process that improves bone tissue transparency prior to light-sheet imaging. The entire protocol, from bone collection to image analysis and quantification, takes about 4 days to complete, thus offering significant improvements over previous methods. This protocol is broadly applicable to the visualization of bone microstructure, bone marrow analysis, vascular and neural network mapping, and the study of signaling molecules in bone development and growth. The protocol requires experience with standard tissue processing and immunostaining techniques, and prior experience in tissue clearing and light-sheet imaging is beneficial but not essential. Key pointsO_LIA protocol for efficient clearing and immunolabeling of intact calcified tissues that enables superfast, high-resolution, and quantitative 3D imaging of various intact bones and teeth. C_LIO_LIThe entire protocol takes only 4 days to complete the comprehensive staining and perfect transparency throughout the intact bones, offering significant improvements over previous methods. C_LI Key referencesBiswas, L. et al. Cell 186, 382-397.e24 (2023): https://doi.org/10.1016/j.cell.2022.12.031

cell biology↗

bioRxiv2026

CryoROLE: describing large inter-domain rotation in single particle cryo-EM

In single particle cryo-EM, analysis of continuous conformational heterogeneity has always been challenging. Both linear and deep learning-based methods treat conformational heterogeneity as perturbations to the consensus average conformation, limiting their capability in analyzing large protein motions. While classic conformational classifications are capable of handling large domain motion, they bin continuous protein dynamics into discrete static substates. Here, we present cryoROLE, a computational tool that extracts the continuous conformational dynamics embedded in the static composite map constructed from multi-body refinement into a landscape of relative orientation between the moving domains. Depicted in real space, the landscape allows intuitive interpretations of domain motion and the population of poses in the conformational space. Applying it to various biological systems reveals hidden conformational dynamics that are relevant to protein functions.

biophysics↗

bioRxiv2026

Recognition Mechanism of Serotonin by a G-Quadruplex-Duplex Hybrid Aptamer

Serotonin is a key neurotransmitter, and aptamer-based tools using the 44-nt Apt44 have been successfully developed for its in vitro and in vivo detection. Nevertheless, the structural basis of recognition by this aptamer remains unclear. Here we report high-resolution NMR structures of Apt38, a 6-nt truncated variant in the third loop of Apt44, in free and serotonin-bound states. Both structures reveal a two-layered antiparallel chair-type G-quadruplex core with three edgewise loops and a terminal duplex, forming a G-quadruplex-duplex hybrid structure. Serotonin binds at the G-quadruplex-duplex junction, stabilized by stacking, electrostatic attraction, hydrogen bonding, and hydrophobic contacts. Apt38 is preorganized for binding, whereas the longer third loop of Apt44 introduces conformational dynamics into the G-quadruplex scaffold, which enables a pronounced binding-triggered conformational switch in PBS buffer, explaining its sensing mechanism. Our work reveals the recognition and sensing mechanism of the serotonin aptamer and provides a framework for aptamer design in serotonin biosensing.

biophysics↗

bioRxiv2026

The peculiar property of pia mater on the prediction of acute subdural hematoma

Acute subdural hematoma (ASDH) is a prevalent injury with high mortality and morbidity, often resulting from bridging vein (BV) disruption secondary to cortical relative motion. As a thin membrane enveloping the brain surface and anchoring BVs, the pia mater is hypothesized to play a critical mechanical role in cortical response and hence ASDH pathogenesis. Finite element (FE) head models are valuable tools to predict ASDH occurrence during impacts. However, the pia mater is often represented as an elastic material in existing FE head models, despite experimental evidence reporting its nonlinear mechanical behavior. In this study, both linear (Youngs modulus of 11.5 MPa) and nonlinear (the stress-strain curve derived from pial tension tests) material models of the pia mater were implemented in one FE head model. The models were subjected to three experimental impact loadings, one of which was known to cause ASDH and two of which were not. Results demonstrated that, across all simulated impacts, the model with nonlinear pia mater properties predicted larger cortical displacements and BV responses than the linear model. For the impact with known ASDH occurrence, the predicted BV strain was 0.17 for the nonlinear model and 0.094 for the linear model, with only the former approaching the reported rupture strain range of the BV-superior sagittal sinus complex (0.29 {+/-} 0.13). These findings verified the mechanical importance of the pia mater in cortical responses and hence the prediction of ASDH, suggesting that conventional linear pia modeling might over-constrain cortical motion, leading to underestimation of BV strain and ASDH risk. The current study supported the adoption of experimentally derived nonlinear pia mater properties in FE head models to improve the reliability of ASDH prediction.

biophysics↗

bioRxiv2026

Pep2Mol: 3D Molecule Generation Targeting Protein-Protein Interfaces with Diffusion Models

Protein-protein interactions (PPIs) are central to biological processes. Designing small molecules that modulate dysregulated PPIs holds strong promise for targeting undruggable proteins. However, existing structure-based drug design approaches focus on well-defined small-molecule binding pockets and struggle to generalize to large, shallow, and chemically complex PPI interfaces. Here, we introduce Pep2Mol, a diffusion-based generative model for 3D molecule design that targets orthosteric PPI sites by explicitly incorporating binding peptides or proteins as structural guidance, moving beyond conventional pocket-conditioned generation. To enable model development and benchmarking, we curate a large-scale, high-quality dataset of 10,956 experimentally resolved protein complex structure pairs, each pairing an orthosteric competitive ligand with a protein binder at overlapping receptor interfaces. Pep2Mol integrates two SE(3)-equivariant graph neural networks that encode protein-ligand and protein-peptide interactions respectively, and fuses these representations via attention-based conditioning to jointly guide the diffusion trajectory. Extensive evaluations demonstrate that Pep2Mol generates chemically valid ligands with state-of-the-art binding affinities, providing a strong foundation for small-molecule inhibitor design against challenging PPI interfaces.

bioinformatics↗

bioRxiv2026

A GATA2-PROX1-FOXC2 regulatory axis integrates shear stress signaling with lymphatic vascular development

The two-hit hypothesis provides a novel framework for incompletely penetrant vascular disorders. Emberger syndrome, caused by heterozygous loss-of-function mutations in mechanosensitive transcription factor (TF) GATA2, is associated with lymphedema in a subset of patients, suggesting that modifiers influence disease development. Heterozygous loss-of-function mutations in mechanosensitive TF FOXC2 also cause lymphedema. Complete deletion of either factor in lymphatic endothelial cells (LECs) causes severe, overlapping defects, including complete loss of lymphatic valves, whereas single heterozygous mutants exhibit milder phenotypes, suggesting genetic buffering. However, whether GATA2 and FOXC2 interact with each other and with shear stress-dependent transcriptional programs remains unclear. Here, we show that Gata2+/-;Foxc2+/- mice develop profound lymphatic vascular defects, including absent lymphatic valves, perinatal lethality, and valve dysfunction, revealing dosage-dependent cooperation between GATA2 and FOXC2. Using a Cre-dependent model of LEC-specific GATA2 overexpression, we found that increased GATA2 dosage downregulated PROX1 and disrupted lymphatic vascular development. ATAC-seq and bulk RNA-seq of shear-exposed human LECs showed that PROX1 governs shear-responsive genes, including KLF2 and KLF4, whereas FOXC2 modulates PROX1-dependent and independent gene subsets. Together, these findings identify PROX1 as a central integrator of shear stress-responsive transcriptional programs and reveal that balanced GATA2 and FOXC2 dosage preserves this network during lymphatic vascular development.

Developmental Biology↗

bioRxiv2026

3D Passive Cavitation Mapping (3D-PCM) with a Large-aperture Planar Array

Urinary stone disease is a common urological condition with increasing incidence, particularly in developed countries. Laser lithotripsy (LL) has become a preferred minimally invasive treatment due to its high precision and low tissue damage. Recent studies suggest that cavitation plays a critical role in stone damage during LL, and three-dimensional passive cavitation mapping (3D-PCM) has emerged as a promising tool for detecting these events. However, clinical translation of 3D-PCM remains challenging due to limitations in imaging depth, field of view (FOV), and procedural compatibility. Here, we present a large-FOV dual-modality imaging system (3D-PCM and B-mode ultrasound) based on a large-aperture planar ultrasound array. Through array optimization and model-based reconstruction, our system achieves an expanded FOV of [~]40 x 40 mm2 at a clinically relevant imaging depth of [~]110 mm, while maintaining high spatial resolution of [~]0.6 mm laterally and [~]0.4 mm axially. In vivo experiments in a porcine model demonstrate that the reconstructed cavitation distribution correlates well with stone damage. Our technology has the potential to provide real-time treatment feedback during LL without disrupting the standard workflow.

bioengineering↗

bioRxiv2026

Improving the detection sensitivity of calcium transients in densely labeled neuronal tissue with pinhole illumination - A low-cost approach

Optical recording from large numbers of neurons is an indispensable technique for studying neuronal ensembles. We use optical sectioning through pinhole illumination to reduce the background fluorescence (F0) and increase the optical signal ({Delta}F/F0) in ex vivo brain slices densely labeled with GCaMP6f, allowing an ordinary fluorescence microscope to capture calcium transients from over 300 individual CA1 neurons - a marked increase compared to ordinary wide field fluorescence illumination. Multiple layers of overlapping neurons can be identified by their locations and the shape in space of their {Delta}F/F0 images. A single pinhole mask was placed at the field stop of a wide field illuminator, and the image of the pinhole was projected onto the tissue by a 20X NA 0.95 water immersion objective (Olympus). This created an illuminated disk with a diameter of [~]200 m and optical sections of hippocampal CA1 pyramidal layer tissue [~]100 m thick. This illumination blocked a large fraction of the F0, which in turn increased the {Delta}F/F0 5-10-fold compared to that of wide field illumination. When putative pyramidal neurons fire sparsely in the brain slice, up to 300 partially superimposed neurons can be identified by their shape and spatial location in the thick ([~]480 m) ex vivo slice in the CA1 area surrounding the pinhole image. The signal-to-noise ratio was adequate even at a low excitation light level of [~]20k photoelectrons per pixel well on the camera, allowing for 3,000 seconds of total recording time without significant bleaching. This pinhole "half confocal" method has created a useful way to sample calcium transient signals in thick tissue with a large population of neurons densely labeled with GCaMP-6f.

neuroscience↗

bioRxiv2026

Flow-Induced Yap/Taz Signaling Balances Endothelial and Hematopoietic Stem Cell Fates

Mechanical forces from blood flow are essential for production of hematopoietic stem and progenitor cells (HSPCs) during embryogenesis, but the molecular mechanisms by which hemodynamic cues are sensed and orchestrate endothelial-to-hematopoietic (EHT) transition remain incompletely defined. We previously identified YAP mechanotransduction as a key integrator of physical forces with EHT. Here we show that hemodynamic forces can activate YAP signaling via the mechanoresponsive ion channel Piezo1 in human iPSC-derived hemogenic endothelium (HE) and zebrafish embryos. Investigation of the Piezo1/YAP axis revealed shared and unique roles of YAP and its paralogue TAZ in EHT. Mechanistically, we find a requirement for the Tead DNA-binding co-factor in YAP/TAZ-dependent control of HSPC number, and note that TAZ uniquely augments transcriptional output of the hematopoietic master regulator Runx1 via direct protein-protein interactions. By comprehensive scRNA-sequencing of YAP/TAZ gain-of-function (GOF) and yap-deficient cells from zebrafish, we reveal that YAP/TAZ promotes HSC production by positively regulating gene programs for hematopoietic self-renewal, cell cycle, and glycolysis-to-oxidative phosphorylation switching, while preventing reversion to endothelial identity. Importantly, comparison of GOF transcriptomes and functional analyses suggest decoupling of metabolic/proliferative and endothelial gene regulatory modules between YAP and TAZ: while either can functionally compensate for loss of the other in EHT, indiscriminate overactivation of TAZ enhances an endothelial program over pro-hematopoietic fate, ultimately blunting progression of HSPC production. Given that hemodynamic cues are integrated simultaneously by arterial and HE cells in embryonic vessels in which EHT occurs, these findings have strong implications for strategies designed to introduce biomechanical cues to in vitro hematopoietic differentiation systems to drive HSC production.

Cell Biology↗