Biology subjects

Liu, Z.

Publications and source records attributed to Liu, Z..

At least 685 records · Page 38Linked to original sources

bioRxiv2022

Cholesterol-deficient T cell membrane-encapsulated STING agonists for tumor-targeted immunotherapy

In recent years, STING agonists have shown promising results in enhancing tumor immunotherapies. Nanoparticle-based tumor targeting delivery of STING agonists is considered as an important way to improve the therapeutic efficacy and reduce the side effects of STING agonists. However, how to escape the clearance to nanoparticles by phagocytes in the blood while maintaining the tumor targeting efficiency of nanoparticles is still a big challenge. Herein, cholesterol-deficient membrane from bioengineered T cells overexpressing PD-1 encapsulated STING agonist SR-717 (a.k.a. COMs) was used to treat melanoma. Nanoparticles coated by these membranes displayed remarkably dropped clearance by monocytes in the blood in both animal and human blood comparing with nanoparticles coated by non-modified T cell membrane, while maintaining the high tumor cell targeting efficiency of COMs. In mice melanoma model, intravenous injected COMs successfully delivered SR-717 to tumor and activated STING pathways and the PD-1 on COMs blocked the up-regulated PD-L1 in tumor cells induced by SR-717. As a result, COMs stimulated strong tumor immune responses to inhibit melanoma recurrence when it combined with photothermal therapy (PTT). In summary, this study developed a highly effective bionic system that integrated STING activation and immunotherapy, and provided a simple and effective strategy to enhance performance of cell membrane-coated delivery systems in vivo.

bioengineering↗

bioRxiv2022

Accurate cell type deconvolution in spatial transcriptomics using a batch effect-free strategy

Sequencing-based spatial transcriptomics (ST) techniques have been groundbreaking in dissecting cell-cell communications within tissues by profiling positional gene expression. However, the most widely used ST technique, Visium Spatial Gene Expression by 10x Genomics (Visium), does not provide single-cell resolution, making it difficult to profile cell type-level information. Many reference-based deconvolution methods have been developed to increase its resolution, but the platform and batch effects between the reference and ST data compromise their accuracy. Here, we propose a new approach, Region-based cell Sorting (ReSort), that generates a pseudo-internal-reference to reduce these platform effects. By simulating ST datasets under various scenarios, we demonstrate that ReSort significantly improves the accuracy of six state-of-the-art reference-based deconvolution methods. Moreover, applying ReSort to a mouse breast cancer tumor bearing both epithelial and mesenchymal clones identifies the spatial differences of immune cells between the clones, providing important insights for understanding the relationship between epithelial-mesenchymal transition and immune infiltration in breast cancer.

bioinformatics↗

bioRxiv2022

Single-Cell Analysis of 5-ALA Intraoperative Labeling Specificity for Glioblastoma

Glioblastoma (GBM) is the most common and aggressive malignant primary brain tumor, and surgical resection is a key part of the standard-of-care. In fluorescence-guided surgery (FGS), fluorophores are used to differentiate tumor tissue from surrounding normal brain. The heme synthesis pathway converts 5-aminolevulinic acid (5-ALA), a fluorogenic substrate used for FGS, to fluorescent protoporphyrin IX (PpIX). The resulting fluorescence is thought to be specific to transformed glioma cells, but this specificity has not been examined at single-cell level. We performed paired single-cell imaging and RNA sequencing of individual cells (SCOPE-seq2) on human GBM surgical specimens with visible PpIX fluorescence from patients who received 5-ALA prior to surgery. SCOPE-seq2 allows us to simultaneously image PpIX fluorescence and unambiguously identify transformed glioma cells from single-cell RNA-seq (scRNA-seq). We observed that 5-ALA treatment results in labeling that is not specific to transformed tumor cells. In cell culture, we further demonstrated that untransformed cells can be labeled by 5-ALA directly or by PpIX secreted from surrounding transformed cells. In acute slice cultures from mouse glioma models, we showed that 5-ALA preferentially labels GBM tumor tissue over non-neoplastic brain tissue, and that this contrast is not due to blood-brain-barrier disruption. Taken together, our findings support the use of 5-ALA as an indicator of GBM tissue, but not as a specific marker of transformed glioma cells.

cancer biology↗

bioRxiv2022

M-Band Wavelet-Based Imputation of scRNA-seq Matrix and Multi-view Clustering of Cell

Wavelet analysis has been recognized as a cutting-edge and promising tool in the fields of signal processing and data analysis. However, application of wavelet-based method in single-cell RNA sequencing (scRNA-seq) data is little known. Here, we present M-band wavelet-based imputation of scRNA-seq matrix and multi-view clustering of cells (WIMC). We applied integration of M-band wavelet analysis and uniform manifold approximation and projection (UMAP) to a panel of single cell sequencing datasets by breaking up the data matrix into a trend (low frequency or low resolution) component and (M-1) fluctuation (high frequency or high resolution) components. We leverage a non-parametric wavelet-based imputation algorithm of sparse data that integrates M-band wavelet transform for recovering dropout events of scRNA-seq datasets. Our method is armed with multi-view clustering of cell types, identity, and functional states, enabling missing cell types visualization and new cell types discovery. Distinct to standard scRNA-seq workflow, our wavelet-based approach is a new addition to resolve the notorious chaotic sparsity of scRNA-seq matrix and to uncover rare cell types with a fine-resolution. Author summaryWe develop M-band wavelet-based imputation of scRNA-seq matrix and multi-view clustering of cells. Our new approach integrates M-band wavelet analysis and UMAP to a panel of single cell sequencing datasets via breaking up the data matrix into a trend (low frequency or low resolution) component and (M - 1) fluctuation (high frequency or high resolution) components. Our method enables us to efficiently impute sparse scRNA-seq data matrix and to examine multi-view clustering of cell types, identity, and functional states, potentializing missing cell types recovery, fine rare cell types discovery, as well as functional cell states exploration.

bioinformatics↗

bioRxiv2022

ExposomeX: Integrative Exposomic Platform Expediates Discovery of "Exposure-Biology-Disease" Nexus

Exposome has become the hotspot of next-generation health studies. To date, there is no available effective platform to standardize the analysis of exposomic data. In this study, we aim to propose one new framework of exposomic analysis and build up one novel integrated platform "ExposomeX" to expediate the discovery of the "Exposure-Biology-Disease" nexus. We have developed 13 standardized modules to accomplish six major functions including statistical learning (E-STAT), exposome database search (E-DB), mass spectrometry data processing (E-MS), meta-analysis (E-META), biological link via pathway integration and protein-protein interaction (E-BIO) and data visualization (E-VIZ). Using ExposomeX, we can effectively analyze the multiple-dimensional exposomics data and investigate the "Exposure-Biology-Disease" nexus by exploring mediation and interaction effects, understanding statistical and biological mechanisms, strengthening prediction performance, and automatically conducting meta-analysis based on well-established literature databases. The performance of ExposomeX has been well validated by re-analyzing two previous multi-omics studies. Additionally, ExposomeX can efficiently help discover new associations, as well as relevant in-depth biological pathways via protein-protein interaction and gene ontology network analysis. In sum, we have proposed a novel framework for standardized exposomic analysis, which can be accessed using both R and online interactive platform (http://www.exposomex.cn/).

bioinformatics↗

bioRxiv2022

Effects of cardiac function alterations on the risk of postoperative thrombotic complications in patients receiving endovascular aortic repair

Chronic heart disease (CHD) is a common comorbidity of patients receiving endovascular aneurysm repair (EVAR) for abdominal aortic aneurysms (AAA). The ventricular systolic function determines the hemodynamic environments in aorta, and thus regulating the formation of postoperative thrombus. However, the explicit relationship between ventricular systolic function and EVAR complication of thrombotic events is unknown. Here, we proposed a three-dimensional numerical model coupled with the lumped-elements heart model, which is capable of simulating thrombus formation in diverse systolic functions. The computational results demonstrate that thrombus tended to form on the interior side of the aorta arch and iliac branches, which is consistent with the four patients post-operative imaging follow-up. In addition, we found that the thrombus formation has negative correlations with the maximum ventricular contractile force (r=-0.2814{+/-}0.1012) and positive correlations with the minimum ventricular contractile force (r=0.238{+/-}0.074), whereas the effect of heart rate (r=-0.0148{+/-}0.1211) on thrombus formation is not significant. In conclusion, changes in ventricular systolic function may alter the risk of thrombotic events after EVAR repair, which could provide insight into the selection of adjuvant therapy strategies for AAA patients with CHD.

bioengineering↗

bioRxiv2022

Mechanisms of ion selectivity and rotor coupling in the bacterial flagellar sodium-driven stator unit

Bacteria swim using a flagellar motor that is powered by stator units. These stator units are energized by an ionic gradient across the membrane, typically proton or sodium. The presumed monodirectional rotation of the stator units allows the bidirectional rotation of the flagellar motor. However, how ion selectivity is attained, how ion transport triggers the directional rotation of the stator unit, and how the stator unit is incorporated into the motor remain largely unclear. Here we have determined by cryo-electron microscopy the structure of the Na+-driven type stator unit PomAB from the gram-negative bacterium Vibrio alginolyticus in both lipidic and detergent environments, at a resolution up to 2.5 [A]. The structure is in a plugged, auto-inhibited state consisting of five PomA subunits surrounding two PomB subunits. The electrostatic potential map uncovers sodium ion binding sites within the transmembrane domain, which together with functional experiments and explicit solvent molecular dynamics simulations, suggest a mechanism for ion translocation and selectivity. Resolved conformational isomers of bulky hydrophobic residues from PomA, in the vicinity of key determinant residues for sodium ion coupling of PomB, prime PomA for clockwise rotation. The rotation is tightly blocked by the trans-mode organization of the PomB plug motifs. The structure also reveals a conformationally dynamic helical motif at the C-terminus of PomA, which we propose regulates the distance between PomA subunit cytoplasmic domains and is involved in stator unit-rotor interaction, concomitant stator unit activation, and torque transmission. Together, our studies provide mechanistic insight for understanding flagellar stator unit ion selectivity and incorporation of the stator units into the motor.

microbiology↗

bioRxiv2022

Epigenomic tomography for probing spatially-defined molecular state in the brain

Spatially-resolved genomic profiling is critical for understanding biology in a spatially ordered organ such as mammalian brain. Single-cell spatial genomic assays were developed recently for this purpose but they remain costly and labor-intensive for examining brain tissues across substantial dimensions and surveying a collection of brain samples. Here we demonstrate a new approach, brain epigenomic tomography, that maps spatial epigenomic variations at the scale of centimeters. We profiled neuronal and glial fractions of mouse neocortex slices with 0.5 mm thickness using a low-input technology. H3K27me3 or H3K27ac features across these slices were grouped into clusters based on their spatial variation patterns. Our approach reveals striking dynamics in frontal and caudal cortex due to kainic acid-induced seizure, linked with transmembrane ion transporter, exocytosis of synaptic vesicles, and secretion of neurotransmitter. Epigenomic tomography provides a powerful and cost-effective tool for profiling and discerning brain samples based on their spatial epigenomes.

genomics↗

bioRxiv2022

Architecture of chloroplast TOC-TIC translocon supercomplex

Chloroplasts rely on the translocon complexes in the outer and inner envelope membranes (termed TOC and TIC, respectively) to import thousands of different nuclear-encoded proteins from the cytosol1-4. While previous studies indicated that the TOC and TIC complexes may assemble into larger supercomplexes5-7, the overall architectures of the TOC-TIC supercomplexes and the mechanism of preprotein translocation are elusive. Here we report the cryo-electron microscopy (cryo-EM) structure of the TOC-TIC supercomplex from Chlamydomonas reinhardtii at an overall resolution of 2.8 [A]. The major subunits of the TOC complex (Toc75, Toc90 and Toc34) and TIC complex (Tic214, Tic20, Tic100 and Tic56), three chloroplast translocon-associated proteins (Ctap3, Ctap4 and Ctap5) and three newly-identified small inner-membrane proteins (Simp1-3) have been located in the supercomplex. As the largest protein, Tic214 traverses the inner membrane, the intermembrane space and the outer membrane, connecting the TOC complex with the TIC proteins. An inositol hexaphosphate (InsP6 or I6P) molecule is located at the Tic214-Toc90 interface and stabilizes their assembly. Moreover, four lipid molecules are located within or above an inner-membrane funnel formed by Tic214, Tic20, Simp1 and Ctap5. Furthermore, multiple potential pathways found in the TOC-TIC supercomplex may support translocation of different substrate preproteins into chloroplasts.

biophysics↗

bioRxiv2022

An HBV mRNA vaccine with favorable virological suppression and superior immunity

BRIEF ABSTRACTHere we report on the development and comprehensive evaluations of an mRNA vaccine for chronic hepatitis B (CHB) treatment. In two different HBV carrier mouse models generated by viral vector-mediated HBV transfection (pAAV-HBV1.2 and rAAV8-HBV1.3), this vaccine demonstrates sufficient and persistent virological suppression, and robust immunogenicity in terms of induction of strong innate immune activation, high-level virus-specific antibodies, memory B cells and T cells. mRNA platform therefore holds prospects for therapeutic vaccine development to combat CHB.

immunology↗

bioRxiv2022

Latent factor in Brain RNA-seq studies reflects cell type and clinical heterogeneity

With the growing availability of Alzheimers disease (AD) transcriptomic data, several studies have nominated new therapeutic targets. However, a major challenge is accounting for latent (hidden) factors which affect the discovery of therapeutic targets. Using unsupervised machine learning, we identified a latent factor in brain tissue, and we validated the factor in AD and normal samples, across multiple studies, and different brain tissues. Moreover, significant metabolic differences were observed due to the latent factor. The latent factor was found to reflect cell-type heterogeneity in the brain and after adjusting for it, we were able to identify new biological pathways. The changes observed at both transcriptomic and metabolomic levels support the importance of identifying any latent factors before pursuing downstream analysis to accurately identify biomarkers.

genomics↗

bioRxiv2022

Tau polarizes an aging transcriptional signature to excitatory neurons and glia

Aging is a major risk factor for Alzheimers disease (AD), and cell-type vulnerability underlies its characteristic clinical manifestations. We have performed longitudinal, single-cell RNA-sequencing in Drosophila with pan-neuronal expression of human tau, which forms AD neurofibrillary tangle pathology. Whereas tau- and aging-induced gene expression strongly overlap (93%), they differ in the affected cell types. In contrast to the broad impact of aging, tau-triggered changes are strongly polarized to excitatory neurons and glia. Further, tau can either activate or suppress innate immune gene expression signatures in a cell type-specific manner. Integration of cellular abundance and gene expression pinpoints Nuclear Factor Kappa B signaling as a potential marker for neuronal vulnerability. We also highlight the conservation of cell type-specific transcriptional patterns between Drosophila and human postmortem brain tissue. Overall, our results create a resource for dissection of dynamic, age-dependent gene expression changes at cellular resolution in a genetically tractable model of tauopathy.

genomics↗

bioRxiv2022

ame-miR-34 modulates the larval body weight and immune response of Apis mellifera workers to Ascosphara apis invasion

MiRNAs are critical regulators of numerous physiological and pathological processes. Ascosphaera apis exclusively infects bee larvae and causes chalkbrood disease. However, the function and mechanism of miRNAs in the bee larval response to A. apis infection is poorly understood. Here, ame-miR-34, a previously predicted miRNA involved in the response of Apis mellifera ligustica larvae to A. apis invasion, was subjected to molecular validation, and overexpression and knockdown were then conducted to explore the regulatory functions of ame-miR-34 in larval body weight and immune response. Stem-loop RT-PCR and Sanger sequencing confirmed the authenticity of ame-miR-34 in the larval gut of A. m. ligustica. RT-qPCR results demonstrated that compared with that in the uninfected larval guts, the expression level of ame-miR-34 was significantly downregulated (P < 0.001) in the guts of A. apis-infected 4-, 5-, and 6-day-old larvae, indicative of the remarkable suppression of host ame-miR-34 due to A. apis infection. In comparison with the corresponding negative control (NC) groups, the expression level of ame-miR-34 in the larval guts in the mimic-miR-34 group was significantly upregulated (P < 0.001), while that in the inhibi- tor-miR-34 group was significantly downregulated (P < 0.01). Similarly, effective overexpression and knockdown of ame-miR-34 were achieved. In addition, the body weights of 5- and 6-day-old larvae were significantly increased compared with those in the mimic-NC group; the weights of 5-day-old larvae in the inhibitor-miR-34 group were significantly decreased in comparison with those in the inhibitor-NC group, while the weights of 4- and 6-day-old larvae in the inhibi- tor-miR-34 group were significantly increased, indicating the involvement of ame-miR-34 in modulating larval body weight. Furthermore, the expression levels of both hsp and abct in the guts of A. apis-infected 4-, 5- and 6-day-old larvae were significantly upregulated after ame-miR-34 overexpression. In contrast, after ame-miR-34 knockdown, the expression levels of the aforementioned two key genes in the A. apis-infected 4-, 5- and 6-day-old larval guts were significantly downregu- lated. Together, the results demonstrated that effective overexpression and knockdown of ame-miR-34 in both noninfected and A. apis-infected A. m. ligustica larval guts could be achieved by the feeding method, and ame-miR-34 exerted a regulatory function in the host immune response to A. apis invasion through positive regulation of the expression of hsp and abct. Our findings not only provide a valuable reference for the functional investigation of bee larval miRNAs but also reveal the regulatory role of ame-miR-34 in A. mellifera larval weight and immune response. Additionally, the results of this study may provide a promising molecular target for the treatment of chalkbrood disease.

molecular biology↗

bioRxiv2022

Trans-eQTL mapping in gene sets identifies network effects of genetic variants

Nearly all trait-associated variants identified in GWAS are non-coding. The cis regulatory effects of these variants have been extensively characterized, but how they impact gene regulation in trans has been the subject of much fewer studies. Mapping trans genetic effects is very challenging because their effect sizes tend to be small and a large multiple testing burden reduces the power to detect them. In addition, read mapping biases can lead to many false positives. To reduce mapping biases and substantially improve power to map trans-eQTLs, we developed a pipeline called trans-PCO, which combines careful read and gene filters with a principal component (PC)-based multivariate association test. Our simulations demonstrate that trans-PCO substantially outperforms existing trans-eQTL mapping methods, including univariate and primary PC-based methods. We applied trans-PCO to two gene expression datasets from whole blood, DGN (N = 913) and eQTLGen (N = 31,684), to identify trans-eQTLs associated with gene co-expression networks and hallmark gene sets representing well-defined biological processes. In total, we identified 14,985 high-quality trans-eSNPs-module pairs associated with 197 co-expression gene modules and biological processes. To better understand the effects of trait-associated variants on gene regulatory networks, we performed colocalization analyses between GWAS loci of 46 complex traits and trans-eQTLs identified in DGN. We highlight several examples where our map of trans effects helps us understand how trait-associated variants impact gene regulatory networks and biological pathways. For example, we found that a locus associated with platelet traits near ARHGEF3 trans-regulates a set of co-expressed genes significantly enriched in the platelet activation pathway. Additionally, six red blood cell trait-associated loci trans-regulate a gene set representing heme metabolism, a crucial process in erythropoiesis. In conclusion, trans-PCO is a powerful and reliable tool that detects trans regulators of cellular pathways and networks, which opens up new opportunities to learn the impact of trait-associated loci on gene regulatory networks.

genetics↗

bioRxiv2022

Distinct structure and gating mechanism in diverse NMDA receptors with GluN2C and GluN2D subunits

N-Methyl-D-Aspartate (NMDA) receptors are essential for many brain functions. These receptors are heterotetramers typically comprising two GluN1 subunits and two GluN2 subunits. The latter could alternate among four subtypes (N2A-N2D) and determine the functional diversity of NMDA receptors1, 2. For example, receptors containing N2C or N2D exhibit 50-fold lower channel open probability (Po) than those containing N2A (ref.3-5). Structures of N2A- and N2B-containing receptors have been extensively characterized, providing molecular basis for understanding NMDA receptor function6-14. Here we report the cryo-EM structures of N1-N2D and N1-N2C di-heterotetramers (di-receptors), and N1-N2A-N2C tri-heterotetramer (tri-receptor) at a resolution up to 3.0 [A]. Structural analysis showed that the bilobate N-terminal domain (NTD) in N2D adopted an intrinsic closed conformation, leading to a compact NTD tetramer in N1-N2D receptor. Functional studies further demonstrated that, in di-receptors containing N2D but not N2A or N2B, crosslinking NTD at the tetrameric interface had no effect on channel activity, while crosslinking ligand-binding domain (LBD) of two N1 protomers significantly elevated Po. Surprisingly, we found that the N1-N2C di-receptors spontaneously oscillated between symmetric and asymmetric conformation. The later one occupied a predominant population, whereby two N2C protomers exhibited distinct conformation. This asymmetry, which was also found to a lesser extent in N1-N2A di-receptor10, was further locked by the binding of an N2C-specific allosteric potentiator PYD-106 to a unique binding pocket between NTD and LBD in only one N2C protomer. Finally, N2A and N2C in the N1-N2A-N2C tri-receptor displayed the conformation close to that found in one protomer of N1-N2A and N1-N2C di-receptors, respectively. These findings provide a comprehensive structural understanding of diverse functional properties of major NMDA receptor subtypes.

neuroscience↗

bioRxiv2022

The photosystem I supercomplex from a primordial green alga Ostreococcus tauri harbors three light-harvesting complex trimers

As a ubiquitous picophytoplankton in the ocean and an early-branching green alga, Ostreococcus tauri is a model prasinophyte species for studying the functional evolution of the light-harvesting systems in photosynthesis. Here, we report the structure and function of the O. tauri photosystem I (PSI) supercomplex in the low light, where it expands its photon-absorbing capacity by assembling with the light-harvesting complexes I (LHCI) and a prasinophyte-specific light-harvesting complex (Lhcp). Its architecture exhibits hybrid features of the plant-type and the green algal-type PSI supercomplexes, consisting of a PSI core, a Lhca1-Lhca4-Lhca2-Lhca3 belt attached on one side and a Lhca5-Lhca6 heterodimer associated on the other side between PsaG and PsaH. Interestingly, nine Lhcp subunits, including one Lhcp1 monomer with a phosphorylated amino-terminal threonine and eight Lhcp2 monomers, oligomerize into three trimers and associate with PSI on the third side between Lhca6 and PsaK. The Lhcp1 phosphorylation and the light-harvesting capacity of PSI were subjected to reversible photoacclimation, suggesting that the formation of OtPSI-LHCI-Lhcp supercomplex is likely due to a state transition-like mechanism induced by light intensity change. Notably, this supercomplex did not exhibit far-red peaks in the 77 K fluorescence spectra, which is possibly due to weak coupling of the chlorophyll a603-a609 pair in OtLhca1-4.

plant biology↗

bioRxiv2022

Patient Clustering and Classification for Vital Organ Failure Using ICD Code with Graph Attention

ObjectiveHeart failure, respiratory failure and kidney failure are three severe organ failures (OF) that have high mortalities and are most prevalent in intensive care units. The objective of this work is to offer insights on OF clustering from the aspects of graph neural network and diagnosis history. MethodsThis paper proposes a neural network-based pipeline to cluster three types of organ failure patients by incorporating embedding pre-train using an ontology graph of International Classification of Diseases (ICD) codes. We employ an autoencoder-based deep clustering architecture jointly trained with a K-means loss, and a non-linear dimension reduction is performed to obtain patient clusters on the MIMIC-III dataset. ResultsThe clustering pipeline shows superior performance on a public-domain image dataset. For MIMIC-III, the model gives two distinct clusters that are related to the severity of the diseases. The learnt ICD embeddings present strong power in identifying the OF type in supervised learning. ConclusionOur proposed pipeline gives stable clusters, however, they do not correspond to the type of OF which indicates these OF share significant hidden characteristics in diagnosis. These clusters can be used to signal possible complications and severity of illness. SignificanceWe are the first to apply an unsupervised approach to offer insights from a biomedical engineering perspective on these three types of organ failure, and publish the pre-trained embeddings for future transfer learning.

bioengineering↗

bioRxiv2022

Integrated transcriptome and lineage analyses reveal novel catecholaminergic cardiomyocytes contributing to the cardiac conduction system in murine heart

Cardiac conduction system (CCS) morphogenesis is essential for correct heart function yet is incompletely understood. Here we established the transcriptional landscape of cell types populating the developing heart by integrating single-cell RNA sequencing and spatial enhanced resolution omics-sequencing (Stereo-seq). Stereo-seq provided a spatiotemporal transcriptomic cell fate map of the murine heart with a panoramic field of view and in situ cellular resolution of the CCS. This led to the identification of a previously unrecognized cardiomyocyte population expressing dopamine beta-hydroxylase (Dbh+-CMs), which is closely associated with the CCS in transcriptomic analyses. To confirm this finding, genetic fate mapping by using DbhCre/Rosa26-tdTomato reporter mouse line was performed with Stereo-seq, RNAscope, and immunohistology. We revealed that Dbh+-derived CMs first emerged in the sinus venosus at E12.5, then populated the atrial and ventricular CCS components at E14.5, with increasing abundance towards perinatal stages. Further tracing by using DbhCFP reporter and DbhCreERT/Rosa26-tdTomato inducible reporter, we confirmed that Dbh+-CMs are mostly abundant in the AVN and ventricular CCS and this persists in the adult heart. By using DbhCre/Rosa26-tdTomato/Cx40-eGFP compound reporter line, we validated a clear co-localization of tdTomato and eGFP signals in both left and right ventricular Purkinje fibre networks. Finally, electrophysiological optogenetic study using cell-type specific Channelrhodopsin2 (ChR2) expression further elucidated that Dbh+-derived CMs form a functional part of the ventricular CCS and display similar photostimulation-induced electrophysiological characteristics to Cx40CreERT/ChR2-tdTomato CCS components. Thus, by utilizing advanced transcriptomic, mouse genetic, and optogenetic functional analyses, our study provides new insights into mammalian CCS development and heterogeneity by revealing novel Dbh+-CMs. HighlightsO_LIStereo-seq provided a spatiotemporal transcriptomic cell fate map of the murine heart with a panoramic field of view and in situ cellular resolution of the CCS. C_LIO_LIEstablished the transcriptional landscape of cell types populating the developing murine heart. C_LIO_LIRevealed previously unreported catecholaminergic cardiomyocyte populations contributing to the developing and mature murine cardiac conduction system. C_LI

cell biology↗