Biology subjects

Lin, R.

Publications and source records attributed to Lin, R..

At least 73 records · Page 4Linked to original sources

bioRxiv2022

The genomes of medicinal skullcaps reveal the polyphyletic origins of clerodane diterpene biosynthesis in the family Lamiaceae.

The presence of anticancer clerodane diterpenoids is a chemotaxonomic marker for the traditional Chinese medicinal plant Scutellaria barbata, although the molecular mechanisms behind clerodane biosynthesis are unknown. Here, we report a high-quality assembly of the 414.98 Mb genome of S. barbata into thirteen pseudochromosomes. Using phylogenomic and biochemical data, we mapped the plastidial metabolism of kaurene (gibberellins), abietane and clerodane diterpenes in three species of the family Lamiaceae (Scutellaria barbata, Scutellaria baicalensis and Salvia splendens), facilitating the identification of genes involved in the biosynthesis of the clerodanes, kolavenol and isokolavenol. We show that clerodane biosynthesis evolved through recruitment and neofunctionalization of genes from gibberellin and abietane metabolism. Despite the assumed monophyletic origin of clerodane biosynthesis which is widespread in species of the Lamiaceae, our data show distinct evolutionary lineages and suggest polyphyletic origins of clerodane biosynthesis in the family Lamiaceae. Our study not only provides significant insights into the evolution of clerodane biosynthetic pathways in the mint family, Lamiaceae, but also will facilitate the elucidation of anticancer clerodanes biosynthesis and future metabolic engineering efforts to increase the production of these high-value chemicals.

plant biology↗

bioRxiv2022

Food for thought: selectivity for food in human ventral visual cortex

Visual cortex contains regions of selectivity for domains of ecological importance. Food is an evolutionarily critical category whose visual heterogeneity may make the identification of selectivity more challenging. We investigate neural responsiveness to food using natural images combined with large-scale human fMRI. Leveraging the improved sensitivity of modern designs and statistical analyses, we identify two food-selective regions in the ventral visual cortex. Our results are robust across 8 subjects from the Natural Scenes Dataset (NSD), multiple independent image sets and multiple analysis methods. We then test our findings of food selectivity in an fMRI "localizer" using grayscale food images. These independent results confirm the existence of food selectivity in ventral visual cortex and help illuminate why earlier studies may have failed to do so. Our identification of food-selective regions stands alongside prior findings of functional selectivity and adds to our understanding of the organization of knowledge within the human visual system.

neuroscience↗

bioRxiv2022

The LEPIS-HuR-TMOD4 axis regulates hepatic cholesterol homeostasis and accelerates atherosclerosis

Long noncoding RNAs (lncRNAs) play an important role in the entire progression of atherosclerosis. In this study, we identified an uncharacterized lncRNA, Liver Expressions by PSRC1 Induce Specifically (LEPIS). The expression of LEPIS and its potential target tropomodulin 4 (TMOD4) in the liver of ApoE-/- mice fed a high-fat diet was increased. An ApoE-/- mouse model with the overexpression of LEPIS or TMOD4 in liver was established, and we found that both LEPIS and TMOD4 increased the burden of atherosclerosis and reduced hepatic cholesterol levels. Further study revealed that LEPIS and TMOD4 affect the expression of genes related to hepatic cholesterol homeostasis, including proprotein convertase subtilisin/kexin type9 (PCSK9) and low-density lipoprotein receptor (LDLR), which are closely related to hypercholesterolemia. Mechanistically, human antigen R (HuR), an RNA-binding protein, was shown to be critical for the regulation of TMOD4 by LEPIS. Further, we found that overexpression of LEPIS promoted the shuttling of HuR from the nucleus to the cytoplasm, enhanced the stability of TMOD4 mRNA, and in turn promoted the expression of TMOD4. In addition, TMOD4 was found to affect intracellular cholesterol levels through PCSK9. These results suggest that the LEPIS-HuR-TMOD4 axis is a potential intervention target for hepatic cholesterol homeostasis and atherosclerosis. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=183 SRC="FIGDIR/small/490400v1_ufig1.gif" ALT="Figure 1"> View larger version (59K): org.highwire.dtl.DTLVardef@c4af60org.highwire.dtl.DTLVardef@d5e7d1org.highwire.dtl.DTLVardef@1667d08org.highwire.dtl.DTLVardef@19abddd_HPS_FORMAT_FIGEXP M_FIG C_FIG

molecular biology↗

bioRxiv2022

Xlnc1DCNN: An Explainable Long Non-Coding RNA Identification Tool

Long non-coding RNAs (lncRNAs) play crucial roles in many biological processes and are implicated in several diseases. With the next-generation sequencing technologies, substantial un-annotated transcripts have been discovered. Classifying unannotated transcripts using biological experiments is more time-consuming and expensive than computational approaches. Several tools for identifying long non-coding RNAs are available. These tools, however, did not explain which features in their tools contributed to the prediction results. Here, we present Xlnc1DCNN, a tool for distinguishing long non-coding RNAs (lncRNAs) from protein-coding transcripts (PCTs) using a one-dimensional convolutional neural network with prediction explanations. The evaluation results of the human test set showed that Xlnc1DCNN outperformed other state-of-the-art tools in terms of accuracy and F1-score. The explanation results revealed that lncRNA transcripts were mainly identified as sequences with no conserved regions or with a region of transmembrane helix while protein-coding transcripts were mostly classified by conserved protein domains or families. The explanation results also conveyed the probably inconsistent annotations among the public databases, lncRNA transcripts which contain protein domains or families, as well as protein-coding transcripts which are nonsense-mediated decay or processed transcripts. Xlnc1DCNN is freely available at https://github.com/cucpbioinfo/Xlnc1DCNN.

bioinformatics↗

bioRxiv2022

Analysis of Sexual vs Asexual Reproduction Using Four Measurements

The prevalence of sexual reproduction has long been an outstanding problem of evolutionary biology. In accordance with the mathematical approach employed by past researchers, we propose a mathematical framework to address this problem. We define and derive four measurements, diversity measure DVM, diffusion measure DFM, optimality measure OPM, and survivability measure SVM to compare sexual reproduction with asexual reproduction. We show that DVM increases exponentially in sexual reproduction, while only linearly in asexual reproduction. Hence, sexual reproduction allows species more opportunity to adapt. We also show that DFM is bounded in sexual reproduction and OPM is inversely related to DFM. Thus, sexual reproduction leads to smaller DFM and hence a larger OPM. We further show that SVM is a monotonic increasing function of OPM. Hence, sexual reproduction is better by virtue of producing a more homogeneous population.

evolutionary biology↗

bioRxiv2022

SARS-CoV-2 impairs interferon production via NSP2-induced repression of mRNA translation

Viruses evade the innate immune response by suppressing the production or activity of cytokines such as type I interferons (IFNs). Here we report the discovery of a novel mechanism by which the SARS-CoV-2 virus co-opts an intrinsic cellular machinery to suppress the production of the key immunostimulatory cytokine IFN-{beta}. We reveal that the SARS-CoV-2 encoded Non-Structural Protein 2 (NSP2) directly interacts with the cellular GIGYF2 protein. This interaction enhances the binding of GIGYF2 to the mRNA cap-binding protein 4EHP, thereby repressing the translation of the Ifnb1 mRNA. Depletion of GIGYF2 or 4EHP significantly enhances IFN-{beta} production, leading to reduced viral infection. Our findings reveal a new target for rescuing the antiviral innate immune response to SARS-CoV-2 and other RNA viruses.

microbiology↗

bioRxiv2021

Improved reference genome annotation of Brassica rapa by PacBio RNA sequencing

The species Brassica rapa includes several important vegetable crops. The draft reference genome of B. rapa ssp. pekinensis was completed in 2011, and it has since been updated twice. The pangenome with structural variations of 18 B. rapa accessions was published in 2021. Although extensive genomic analysis has been conducted on B. rapa, a comprehensive genome annotation including gene structure, alternative splicing events, and non-coding genes is still lacking. Therefore, we used the Pacific Biosciences (PacBio) single-molecular long-read technology to improve gene models and produced the annotated genome version 3.5. In total, we obtained 753,041 full-length non-chimeric (FLNC) reads and collapsed these into 92,810 non-redundant consensus isoforms, capturing 48% of the genes annotated in the B. rapa reference genome annotation v3.1. Based on the isoform data, we identified 830 novel protein-coding genes that were missed in previous genome annotations, defined the UTR regions of 20,340 annotated genes and corrected 886 wrongly-spliced genes. We also identified 28,564 alternative splicing (AS) events and 1,480 long non-coding RNAs (lncRNAs). We produced a relatively complete and high-quality reference transcriptome for B. rapa that can facilitate further functional genomic research.

plant biology↗

bioRxiv2021

SARS-COV-2 induced Diarrhea is inflammatory, Ca2+ Dependent and involves activation of calcium activated Cl channels

Diarrhea occurs in 2-50% of cases of COVID-19 ([~]8% is average across series). The diarrhea does not appear to account for the disease mortality and its contribution to the morbidity has not been defined, even though it is a component of Long Covid or post-infectious aspects of the disease. Even less is known about the pathophysiologic mechanism of the diarrhea. To begin to understand the pathophysiology of COVID-19 diarrhea, we exposed human enteroid monolayers obtained from five healthy subjects and made from duodenum, jejunum, and proximal colon to live SARS-CoV-2 and virus like particles (VLPs) made from exosomes expressing SARS-CoV-2 structural proteins (Spike, Nucleocapsid, Membrane and Envelope). Results: 1) Live virus was exposed apically for 90 min, then washed out and studied 2 and 5 days later. SARS-Cov-2 was taken up by enteroids and live virus was present in lysates and in the apical>>basolateral media of polarized enteroids 48 h after exposure. This is the first demonstration of basolateral appearance of live virus after apical exposure. High vRNA concentration was detected in cell lysates and in the apical and basolateral media up to 5 days after exposure. 2) Two days after viral exposure, cytokine measurements of media showed significantly increased levels of IL-6, IL-8 and MCP-1. 3) Two days after viral exposure, mRNA levels of ACE2, NHE3 and DRA were reduced but there was no change in mRNA of CFTR. NHE3 protein was also decreased. 4) Live viral studies were mimicked by some studies with VLP exposure for 48 h. VLPs with Spike-D614G bound to the enteroid apical surface and was taken up; this resulted in decreased mRNA levels of ACE2, NHE3, DRA and CFTR. 4) VLP effects were determined on active anion secretion measured with the Ussing chamber/voltage clamp technique. S-D614G acutely exposed to apical surface of human ileal enteroids did not alter the short-circuit current (Isc). However, VLPS-D614G exposure to enteroids that were pretreated for [~]24 h with IL-6 plus IL-8 induced a concentration dependent increase in Isc indicating stimulated anion secretion, that was delayed in onset by [~]8 min. The anion secretion was inhibited by apical exposure to a specific calcium activated Cl channel (CaCC) inhibitor (AO1) but not by a specific CFTR inhibitor (BP027); was inhibited by basolateral exposure to the K channel inhibit clortimazole; and was prevented by pretreatment with the calcium buffer BAPTA-AM. 5) The calcium dependence of the VLP-induced increase in Isc was studied in Caco-2/BBe cells stably expressing the genetically encoded Ca2+ sensor GCaMP6s. 24 h pretreatment with IL-6/IL-8 did not alter intracellular Ca2+. However, in IL-6/IL-8 pretreated cells, VLP S-D614G caused appearance of Ca2+waves and an overall increase in intracellular Ca2+ with a delay of [~]10 min after VLP addition. We conclude that the diarrhea of COVID-19 appears to an example of a calcium dependent inflammatory diarrhea that involves both acutely stimulated Ca2+ dependent anion secretion (stimulated Isc) that involves CaCC and likely inhibition of neutral NaCl absorption (decreased NHE3 protein and mRNA and decreased DRA mRNA).

physiology↗

bioRxiv2021

EgGLUT1 is crucial for the viability of larvae of Echinococcus granulosus sensus lato by involving its glucose uptake

Cystic echinococcosis (CE) is a zoonotic parasitic disease caused by infection with the larvae of Echinococcus granulosus sensu lato (s.l.) cluster. It is urgent to identify novel drug targets and develop new drug candidates against CE. Glucose transporter 1 (GLUT1) is mainly responsible for the transmembrane transport of glucose to maintain its constant cellular availability and is a recent research hotspot as a drug target in various diseases. However, presence and role of GLUT1 in E. granulosus s.l. (EgGLTU1) was unknown. In this study, we cloned a conserved GLUT1 homology gene (named EgGLUT1-ss) from E. granulosus sensu stricto (s.s.) and found EgGLUT1-ss was crucial for glucose uptake of the protoscoleces of E. granulosus s.s.. WZB117, a GLUT1 inhibitor, inhibited glucose uptake of E. granulosus s.s. and the viability of the metacestode in vitro. In addition, WZB117 showed potent therapeutic activity in E. granulosus s.s.-infected mice: a 10 mg/kg dose of WZB117 significantly reduced the number and weight of parasite cysts as well as the reference drug, albendazole. Our data have defined EgGLUT1 as a key E. granulosus s.l. vulnerability target, involved in its glucose uptake from the host; this opens a new avenue to identify drugs with an ideal activity profile for the treatment of CE.

microbiology↗

bioRxiv2021

Selection and Validation of Reference Genes Desirable for Gene Expression Analysis by qRT-PCR on Seed Germination of Castanea henryi

Seed germination is the beginning of the plants life cycle, and seed biology is one of the most extensively researched areas in plant physiology, however, Castanea henryi as an important seed plant, the stable internal reference gene during germination is not clear. In this study, seven candidate genes (TUA, TUB, TIF, UBC, RPL21, RPL30, RPL34) were screened out from transcriptome data, we analyzed the expression of seven candidate reference genes in C. henryi at different germination stages with RT-qPCR, and using common algorithms including NormFinder, geNorm and BestKeeper to evaluate the candidate genes stability. The results showed that RPL34 and RPL30 were selected as the most stable genes by NormFinder; TIF was the most stable gene identified by BestKeeper; RPL34 and RPL21 were the most stable genes ranked by geNorm, and TUB was the most unstable gene identified by all of the three software. The RPL34 gene was used as the reference gene, to detected the expression trend of two starch synthetase genes SS1 and SS2 during germination by RT-qPCR, the results of RT-qPCR and transcriptome sequencing were basically consistent, which verified the stability of RPL34 candidate gene. Our result is not only showed functional genes for germination of C. henryi seeds and provide useful guidelines for the selection of reliable reference genes for the normalization of RT- qPCR data for germination of seed plants.

molecular biology↗

bioRxiv2020

TRPM2 promotes pancreatic cancer by PKC/MAPK pathway

BackgroundThe mechanism of pancreatic cancer(PA) is not fully understanded. In our last report, TRPM2 plays a promising role in pancreatic cancer. However, the mechanism of TRPM2 is still unknown in this dismal disease. This study was designed to investigate the role and mechanism of TRPM2 in pancreatic cancer. MethodsTRPM2 overexpressed and siRNA plasmid were created and transfected with pancreatic cancer cell line(BxPC-3) to construct the cell model. We employed CCK-8, Transwell, scratch wound, and nude mice tumor bearing model to investigate the role of TRPM2 in pancreatic cancer. Besides, we collected the clinical data, tumor tissue sample(TT) and para-tumor sample(TP) from the pancreatic cancer patients treated in our hospital. We analyzed the mechanism of TRPM2 in pancreatic cancer by transcriptome analysis, Westernblot, and PCR. ResultsOverexpressed TRPM2 could promote pancreatic cancer in proliferation, migration, and invasion ability in no matter the cell model or nude mice tumor bearing model. TRPM2 level is highly negative correlated to the overall survival and progression-free survival time in PA patients, however, it is significantly increased in PA tissue as the tumor stage increases. The transcriptome analysis, GSEA analysis, Westernblot, and PCR results indicates TRPM2 is highly correlated with PKC/MAPK pathways. ConclusionTRPM2 could directly activate PKC by calcium or indirectly activate PKC{varepsilon} and PKC{delta} by increased DAG in PC, which promote PC by downstream MAPK/MEK pathway activation.

cancer biology↗

bioRxiv2020

Full three-dimensional imaging deep through multicellular thick samples with subcellular resolution by structured illumination microscopy and adaptive optics

Structured Illumination Microscopy enables live imaging with resolutions of ~120 nm. Unfortunately, optical aberrations can lead to loss of resolution and artifacts in Structured Illumination Microscopy rendering the technique unusable in samples thicker than a single cell. Here we report on the combination of Adaptive Optics and Structured Illumination Microscopy enabling imaging with 140 nm lateral and 585 nm axial resolution in tissue culture cells, C. elegans, and rice blast fungus. We demonstrate that AO improves resolution and reduces artifacts, making full 3D SIM possible in thicker samples.

cell biology↗

bioRxiv2020

Multiplexed drug testing of tumor slices using a microfluidic platform

Current methods to assess the drug response of individual human cancers are often inaccurate, costly, or slow. Functional approaches that rapidly and directly assess the response of patient cancer tissue to drugs or small molecules offer a promising way to improve drug testing, and have the potential to identify the best therapy for individual patients. We developed a digitally-manufactured microfluidic platform for multiplexed drug testing of intact cancer slice cultures, and demonstrate the use of this platform to evaluate drug responses in slice cultures from human glioma xenografts and patient tumor biopsies. This approach retains much of the tissue microenvironment and can provide results rapidly enough, within days of surgery, to guide the choice of effective initial therapies. Our results establish a useful preclinical platform for cancer drug testing and development with the potential to improve cancer personalized medicine.

bioengineering↗

bioRxiv2019

Endothelial JAK2V617F mutation leads to thrombosis, vasculopathy, and cardiomyopathy in a murine model of myeloproliferative neoplasm

RationalThe myeloproliferative neoplasms (MPNs) are clonal hematological malignancies characterized by hematopoietic stem cell expansion and overproduction of mature blood cells. Cardiovascular complications are the leading cause of morbidity and mortality in patients with MPNs. The acquired kinase mutation JAK2V617F plays a central role in these disorders. Mechanisms responsible for cardiovascular dysfunction in MPNs are not fully understood, limiting the effectiveness of current treatment. ObjectiveVascular endothelial cells (ECs) play critical roles in the regulation of hemostasis and thrombosis. ECs carrying the JAK2V617F mutation can be detected in patients with MPNs. The goal of this study was to test the hypothesis that the JAK2V617F mutation alters endothelial function to promote cardiovascular complications in patients with MPNs. Methods and ResultsWe employed murine models of MPN in which the JAK2V617F mutation is expressed in specific cell lineages. When JAK2V617F is expressed in both blood cells and vascular ECs, the mice developed MPN and spontaneous, age-related dilated cardiomyopathy with an increased risk of sudden death as well as a prothrombotic and vasculopathy phenotype on histology evaluation. We showed that JAK2V617F-mutant ECs are required for this cardiovascular disease phenotype and the mutation can alter endothelial cell function. Finally, in a more therapeutically oriented approach, we demonstrated that transplantation with wild-type donor marrow cells can improve cardiac function by reversing the left ventricle remodeling process in this JAK2V617F-positive MPN murine model. ConclusionsThese findings suggest that the JAK2V617F mutation alters vascular endothelial function to promote cardiovascular complications in MPNs. Therefore, targeting the MPN vasculature represents a promising new therapeutic strategy for patients with MPNs.

molecular biology↗

bioRxiv2019

Saak Transform-Based Machine Learning for Light-Sheet Imaging of Cardiac Trabeculation

Recent advances in light-sheet fluorescence microscopy (LSFM) enable 3-dimensional (3-D) imaging of cardiac architecture and mechanics in toto. However, segmentation of the cardiac trabecular network to quantify cardiac injury remains a challenge. We hereby employed \"subspace approximation with augmented kernels (Saak) transform\" for accurate and efficient quantification of the light-sheet image stacks following chemotherapy-treatment. We established a machine learning framework with augmented kernels based on the Karhunen-Loeve Transform (KLT) to preserve linearity and reversibility of rectification. The Saak transform-based machine learning enhances computational efficiency and obviates iterative optimization of cost function needed for neural networks, minimizing the number of training data sets to three 2-D slices for segmentation in our scenario. The integration of forward and inverse Saak transforms serves as a light-weight module to filter adversarial perturbations and reconstruct estimated images, salvaging robustness of existing classification methods. The accuracy and robustness of the Saak transform are evident following the tests of dice similarity coefficients and various adversary perturbation algorithms, respectively. The addition of edge detection further allows for quantifying the surface area to volume ratio (SVR) of the myocardium in response to chemotherapy-induced cardiac remodeling. The combination of Saak transform, random forest, and edge detection augments segmentation efficiency by 20-fold as compared to manual processing; thus, establishing a robust framework for post light-sheet imaging processing, creating a data-driven machine learning for 3-D quantification of cardiac ultra-structure.

bioengineering↗

bioRxiv2019

Widespread arginine phosphorylation in human cells - a novel protein PTM revealed by mass spectrometry

Arginine phosphorylation (pArg) is recently discovered as a ubiquitous protein N- phosphorylation in bacteria. However, its prevalence and roles in mammalian cells remain largely unknown due to the lack of established workflow and the inherent lability of the phosphoramidate (P-N) bond. Emerging evidence suggests that N-phosphorylation may extensively exist in eukaryotes and play crucial roles. We report an experimental phosphoproteomic workflow, which for the first time allowed to reveal the widespread occurrence of pArg in human cells by mass spectrometry. By virtue of this approach, we identified 152 high-confidence pArg sit]es derived from 118 proteins. Remarkably, the discovered phosphorylation motif and gene ontology of pArg hint a possible cellular function of arginine phosphorylation by regulating the favorability of propeptide convertase substrate. The generated extensive data set should enable a better understanding of the biological functions of eukaryotic pArg in the future.\n\n\n\nO_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=37 SRC=\"FIGDIR/small/725291v1_ufig1.gif\" ALT=\"Figure 1\">\nView larger version (13K):\norg.highwire.dtl.DTLVardef@77b2d0org.highwire.dtl.DTLVardef@7c76f4org.highwire.dtl.DTLVardef@1658d6borg.highwire.dtl.DTLVardef@27a2e2_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗

bioRxiv2019

CD155/CD96 promotes immunosuppression in lung adenocarcinoma (LUAD)

Lung adenocarcinoma (LUAD) remains one of the leading causes of death in patients with cancer. The association of CD155 with CD96 transmits an inhibitory signal and suppresses antitumor immune response. This study investigates the effect of CD155/CD96 on immune suppression in LUAD. We demonstrate that LUAD patients with high CD155 expression suffer from immune suppression and experience a poor prognosis, which coincides with an inhibited AKT-mTOR signaling pathway in CD8 T cells and subsequently up-regulated CD96 expression. Moreover, the inhibition effect can be reversed by CD96 blocking antibody. High CD155 expression inhibited the release of IFN{gamma} from CD8 cells. Moreover, Blocking CD96 restored IFN{gamma} production in CD8 T cells and neutralized the inhibition of IFN{gamma} production in CD8 T cells mediated by CD155. Animal experiments showed that CD155-mediated LUAD growth might depend on its suppression antitumor immune response in the tumor microenvironment in PDX mice. In conclusion, our results suggest that LUAD cells suppress antitumor immune response in the tumor microenvironment through CD155/CD96. CD155/CD96 could be a potential therapeutic target for LUAD patients.\n\nAbbreviationsLUAD: lung adenocarcinoma; IFN{gamma}: interferon gamma; PDX: patient-derived xenograft; NSCLC: non-small cell lung cancer; PRR: poliovirus receptor-related; MDSCs: myeloid-derived suppressor cells; PRR: poliovirus receptor-related; STR: short tandem repeat; IRS: immunoreactive score; SI: staining intensity; PP: percentage of positive cells; RT-PCR: reverse transcription-polymerase chain reaction; PBS: phosphate-buffered saline; PBMCs: peripheral blood mononuclear cells; SDS-PAGE: sodium dodecyl sulfate-polyacrylamide gel electrophoresis; rCD155: recombinant human CD155; LUAD cells: lung adenocarcinoma cells; TILs: tumor-infiltrating lymphocytes; GzmB: granzyme B; IL-2 (Interleukin-2); TNF- : tumor necrosis factor-alpha; PI: propidium Iodide; PDX: patient-derived xenograft; TIGIT: T cell immunoreceptor with Igand ITIM domains; WBC: white blood cells; MFI: mean fluorescence intensity; HPF: high power field

cancer biology↗

bioRxiv2019

Serotonin Neurons in the Dorsal and Medial Raphe Nuclei: from Single-Cell Transcriptomes to Whole-Brain Projections

Serotonin neurons of the dorsal and medial raphe nuclei (DR and MR) collectively innervate the entire forebrain and midbrain, modulating diverse physiology and behavior. To gain a fundamental understanding of their molecular heterogeneity, we used plate-based single-cell RNA-sequencing to generate a comprehensive dataset comprising eleven transcriptomically distinct serotonin neuron clusters. Systematic in situ hybridization mapped specific clusters to the principal DR, caudal DR, or MR. These transcriptomic clusters differentially express a rich repertoire of neuropeptides, receptors, ion channels, and transcription factors. We generated novel intersectional viral-genetic tools to access specific subpopulations. Whole-brain axonal projection mapping revealed that DR serotonin neurons co-expressing vesicular glutamate transporter-3 preferentially innervate the cortex, whereas those co-expressing thyrotropin-releasing hormone innervate subcortical regions in particular the hypothalamus. Reconstruction of 50 individual DR serotonin neurons revealed segregated axonal projection patterns at the single-cell level. Together, these results provide a molecular foundation of the heterogenous serotonin neuronal phenotypes.

neuroscience↗