bioRxiv : the preprint server for biology
Su, Y;Xu, J;Zhu, Z;Yu, H;Nudell, V;Dash, B;Moya, EA;Ye, L;Nimmerjahn, A;Sun, X;
PMID: 36778350 | DOI: 10.1101/2023.02.04.527145
Chronic exposure of the lung to irritants such as allergen is a primary cause of asthma characterized by exaggerated airway constriction, also called hyperreactivity, which can be life-threatening. Aside from immune cells, vagal sensory neurons are important for airway hyperreactivity 1â€"4 . However, the identity and signature of the downstream nodes of this adaptive circuit remains poorly understood. Here we show that a single population of Dbh + neurons in the nucleus of the solitary tract (nTS) of the brainstem, and downstream neurons in the nucleus ambiguous (NA), are both necessary and sufficient for chronic allergen-induced airway hyperreactivity. We found that repeated exposures of mice to inhaled allergen activates nTS neurons in a mast cell-, interleukin 4 (IL-4)- and vagal nerve-dependent manner. Single-nucleus RNA-seq of the nTS at baseline and following allergen challenges reveals that a Dbh + population is preferentially activated. Ablation or chemogenetic inactivation of Dbh + nTS neurons blunted, while chemogenetic activation promoted hyperreactivity. Viral tracing indicates that Dbh + nTS neurons, capable of producing norepinephrine, project to the NA, and NA neurons are necessary and sufficient to relay allergen signals to postganglionic neurons that then directly drive airway constriction. Focusing on transmitters, delivery of norepinephrine antagonists to the NA blunted allergen-induced hyperreactivity. Together, these findings provide molecular, anatomical and functional definitions of key nodes of a canonical allergen response circuit. The knowledge opens the possibility of targeted neural modulation as an approach to control refractory allergen-induced airway constriction.
Kaneko, K;Sato, Y;Uchino, E;Toriu, N;Shigeta, M;Kiyonari, H;Endo, S;Fukuma, S;Yanagita, M;
PMID: 35644281 | DOI: 10.1016/j.kint.2022.04.026
Erythropoietin (Epo) is produced by a subpopulation of resident fibroblasts in the healthy kidney. We have previously demonstrated that, during kidney fibrosis, kidney fibroblasts including Epo-producing cells transdifferentiate into myofibroblasts and lose their Epo-producing ability. However, it remains unclear whether Epo-producing cells survive and transform into myofibroblasts during fibrosis because previous studies did not specifically label Epo-producing cells in pathophysiological conditions. Here, we generated EpoCreERT2/+ mice, a novel mouse strain that enables labeling of Epo-producing cells at desired time points and examined the behaviors of Epo-producing cells under pathophysiological conditions. Lineage -labeled cells that were producing Epo when labeled were found to be a small subpopulation of fibroblasts located in the interstitium of the kidney, and their number increased during phlebotomy-induced anemia. Around half of lineage-labeled cells expressed Epo mRNA, and this percentage was maintained even 16 weeks after recombination, supporting the idea that a distinct subpopulation of cells with Epo-producing ability makes Epo repeatedly. During fibrosis caused by ureteral obstruction, EpoCreERT2/+ -labeled cells were found to transdifferentiate into myofibroblasts with concomitant loss of Epo-producing ability, and their numbers and the proportion among resident fibroblasts increased during fibrosis, indicating their high proliferative capacity. Finally, we confirmed that EpoCreERT2/+-labeled cells that lost their Epo-producing ability during fibrosis regained their ability after kidney repair due to relief of the ureteral obstruction. Thus, our analyses have revealed previously unappreciated characteristic behaviors of Epo-producing cells, which had not been clearly distinguished from those of resident fibroblasts.
Chevée M, Robertson JJ, Cannon GH, Brown SP, Goff LA.
PMID: 29320739 | DOI: 10.1016/j.celrep.2017.12.046
Single-cell RNA sequencing has generated catalogs of transcriptionally defined neuronal subtypes of the brain. However, the cellular processes that contribute to neuronal subtype specification and transcriptional heterogeneity remain unclear. By comparing the gene expression profiles of single layer 6 corticothalamic neurons in somatosensory cortex, we show that transcriptional subtypes primarily reflect axonal projection pattern, laminar position within the cortex, and neuronal activity state. Pseudotemporal ordering of 1,023 cellular responses to sensory manipulation demonstrates that changes in expression of activity-induced genes both reinforced cell-type identity and contributed to increased transcriptional heterogeneity within each cell type. This is due to cell-type biased choices of transcriptional states following manipulation of neuronal activity. These results reveal that axonal projection pattern, laminar position, and activity state define significant axes of variation that contribute both to the transcriptional identity of individual neurons and to the transcriptional heterogeneity within each neuronal subtype.
O'Toole, A;Mohamed, F;Zhang, J;Brown, C;
| DOI: 10.2139/ssrn.4199232
To detail early tissue distribution and innate immune response to rabbit hemorrhagic disease virus 2 (RHDV2), 13 rabbits were orally ( Oryctolagus cuniculus ) inoculated with liver homogenate made from a feral rabbit that succumbed to RHDV2 during the 2020 outbreak in Oregon, USA. Rabbits were monitored regularly, with euthanasia and collection of tissues and swabs, at 12, 24, 36, 48, 96, and 144 hours post inoculation. Livers from these rabbits were positive by RT-rtPCR for presence of the virus. Using RNAscope for viral and replicative intermediates, rabbits had detectable viral genomic RNA at each time point, initially within the gastrointestinal tract, then in the liver by 36 hours post inoculation. Also using RNAscope, there were increasing amounts of mRNA coding for TNF-α, IL-6, and IL-1β within the liver and spleen through 48 hours post inoculation. The results of this study aided our understanding of the local innate immune response to RHDV2, as well as aspects of pathogenesis.
François A, Low SA, Sypek EI, Christensen AJ, Sotoudeh C, Beier KT, Ramakrishnan C, Ritola KD, Sharif-Naeini R, Deisseroth K, Delp SL, Malenka RC, Luo L, Hantman AW, Scherrer G.
PMID: 28162807 | DOI: 10.1016/j.neuron.2017.01.008
Pain thresholds are, in part, set as a function of emotional and internal states by descending modulation of nociceptive transmission in the spinal cord. Neurons of the rostral ventromedial medulla (RVM) are thought to critically contribute to this process; however, the neural circuits and synaptic mechanisms by which distinct populations of RVM neurons facilitate or diminish pain remain elusive. Here we used in vivo opto/chemogenetic manipulations and trans-synaptic tracing of genetically identified dorsal horn and RVM neurons to uncover an RVM-spinal cord-primary afferent circuit controlling pain thresholds. Unexpectedly, we found that RVM GABAergic neurons facilitate mechanical pain by inhibiting dorsal horn enkephalinergic/GABAergic interneurons. We further demonstrate that these interneurons gate sensory inputs and control pain through temporally coordinated enkephalin- and GABA-mediated presynaptic inhibition of somatosensory neurons. Our results uncover a descending disynaptic inhibitory circuit that facilitates mechanical pain, is engaged during stress, and could be targeted to establish higher pain thresholds.
Albisetti, GW;Ganley, RP;Pietrafesa, F;Werynska, K;Magalhaes de Sousa, M;Sipione, R;Scheurer, L;Bösl, MR;Pelczar, P;Wildner, H;Zeilhofer, HU;
PMID: 36323322 | DOI: 10.1016/j.neuron.2022.10.008
Proper sensing of ambient temperature is of utmost importance for the survival of euthermic animals, including humans. While considerable progress has been made in our understanding of temperature sensors and transduction mechanisms, the higher-order neural circuits processing such information are still only incompletely understood. Using intersectional genetics in combination with circuit tracing and functional neuron manipulation, we identified Kcnip2-expressing inhibitory (Kcnip2GlyT2) interneurons of the mouse spinal dorsal horn as critical elements of a neural circuit that tunes sensitivity to cold. Diphtheria toxin-mediated ablation of these neurons increased cold sensitivity without affecting responses to other somatosensory modalities, while their chemogenetic activation reduced cold and also heat sensitivity. We also show that Kcnip2GlyT2 neurons become activated preferentially upon exposure to cold temperatures and subsequently inhibit spinal nociceptive output neurons that project to the lateral parabrachial nucleus. Our results thus identify a hitherto unknown spinal circuit that tunes cold sensitivity.
Mu, R;Tang, S;Han, X;Wang, H;Yuan, D;Zhao, J;Long, Y;Hong, H;
PMID: 35649349 | DOI: 10.1016/j.celrep.2022.110882
Generalization of visual aversion is a critical function of the brain that supports survival, but the underlying neurobiological mechanisms are unclear. We establish a rapid generalization procedure for inducing visual aversion by dynamic stripe images. By using fiber photometry, apoptosis, chemogenetic and optogenetic techniques, and behavioral tests, we find that decreased cholinergic neurons' activity in the medial septum (MS) leads to generalization loss of visual aversion. Strikingly, we identify a projection from MS cholinergic neurons to the medial habenula (MHb) and find that inhibition of the MS→MHb cholinergic circuit disrupts aversion-generalization formation while its continuous activation disrupts subsequent extinction. Further studies show that MS→MHb cholinergic projections modulate the generalization of visual aversion possibly via M1 muscarinic acetylcholine receptors (mAChRs) of downstream neurons coreleasing glutamate and acetylcholine. These findings reveal that the MS→MHb cholinergic circuit is a critical node in aversion-generalization formation and extinction and potentially provides insight into the pathogenesis of affective disorders.
Gajewski, T;Rouhani, S;Trujillo, J;Pyzer, A;Yu, J;Fessler, J;Cabanov, A;Higgs, E;Cron, K;Zha, Y;Lu, Y;Bloodworth, J;Abasiyanik, M;Okrah, S;Flood, B;Hatogai, K;Leung, M;Pezeshk, A;Kozloff, L;Reschke, R;Strohbehn, G;Chervin, CS;Kumar, M;Schrantz, S;Madariaga, ML;Beavis, K;Yeo, KT;Sweis, R;Segal, J;Tay, S;Izumchenko, E;Mueller, J;Chen, L;
PMID: 34845442 | DOI: 10.21203/rs.3.rs-1083825/v1
The mechanisms explaining progression to severe COVID-19 remain poorly understood. It has been proposed that immune system dysregulation/over-stimulation may be implicated, but it is not clear how such processes would lead to respiratory failure. We performed comprehensive multiparameter immune monitoring in a tightly controlled cohort of 128 COVID-19 patients, and used the ratio of oxygen saturation to fraction of inspired oxygen (SpO2 / FiO2) as a physiologic measure of disease severity. Machine learning algorithms integrating 139 parameters identified IL-6 and CCL2 as two factors predictive of severe disease, consistent with the therapeutic benefit observed with anti-IL6-R antibody treatment. However, transcripts encoding these cytokines were not detected among circulating immune cells. Rather, in situ analysis of lung specimens using RNAscope and immunofluorescent staining revealed that elevated IL-6 and CCL2 were dominantly produced by infected lung type II pneumocytes. Severe disease was not associated with higher viral load, deficient antibody responses, or dysfunctional T cell responses. These results refine our understanding of severe COVID-19 pathophysiology, indicating that aberrant cytokine production by infected lung epithelial cells is a major driver of immunopathology. We propose that these factors cause local immune regulation towards the benefit of the virus.
Chen, W;Liu, X;Li, W;Shen, H;Zeng, Z;Yin, K;Priest, JR;Zhou, Z;
PMID: 34569705 | DOI: 10.15252/embr.202152389
The migratory cardiac neural crest cells (CNCCs) contribute greatly to cardiovascular development. A thorough understanding of the cell lineages, developmental chronology, and transcriptomic states of CNCC derivatives during normal development is essential for deciphering the pathogenesis of CNCC-associated congenital anomalies. Here, we perform single-cell transcriptomic sequencing of 34,131 CNCC-derived cells in mouse hearts covering eight developmental stages between E10.5 and P7. We report the presence of CNCC-derived mural cells that comprise pericytes and microvascular smooth muscle cells (mVSMCs). Furthermore, we identify the transition from the CNCC-derived pericytes to mVSMCs and the key regulators over the transition. In addition, our data support that many CNCC derivatives had already committed or differentiated to a specific lineage when migrating into the heart. We explore the spatial distribution of some critical CNCC-derived subpopulations with single-molecule fluorescence in situ hybridization. Finally, we computationally reconstruct the differentiation path and regulatory dynamics of CNCC derivatives. Our study provides novel insights into the cell lineages, developmental chronology, and regulatory dynamics of CNCC derivatives during development.
Zhao, Q;Yu, CD;Wang, R;Xu, QJ;Dai Pra, R;Zhang, L;Chang, RB;
PMID: 35296859 | DOI: 10.3760/cma.j.cn112151-20210719-00516
Interoception, the ability to timely and precisely sense changes inside the body, is critical for survival1-4. Vagal sensory neurons (VSNs) form an important body-to-brain connection, navigating visceral organs along the rostral-caudal axis of the body and crossing the surface-lumen axis of organs into appropriate tissue layers5,6. The brain can discriminate numerous body signals through VSNs, but the underlying coding strategy remains poorly understood. Here we show that VSNs code visceral organ, tissue layer and stimulus modality-three key features of an interoceptive signal-in different dimensions. Large-scale single-cell profiling of VSNs from seven major organs in mice using multiplexed projection barcodes reveals a 'visceral organ' dimension composed of differentially expressed gene modules that code organs along the body's rostral-caudal axis. We discover another 'tissue layer' dimension with gene modules that code the locations of VSN endings along the surface-lumen axis of organs. Using calcium-imaging-guided spatial transcriptomics, we show that VSNs are organized into functional units to sense similar stimuli across organs and tissue layers; this constitutes a third 'stimulus modality' dimension. The three independent feature-coding dimensions together specify many parallel VSN pathways in a combinatorial manner and facilitate the complex projection of VSNs in the brainstem. Our study highlights a multidimensional coding architecture of the mammalian vagal interoceptive system for effective signal communication.
Huang, XT;Li, T;Li, T;Xing, S;Tian, JZ;Ding, YF;Cai, SL;Yang, YS;Wood, C;Yang, JS;Yang, WJ;
PMID: 36516755 | DOI: 10.1016/j.celrep.2022.111796
Intestinal epithelial replenishment is fueled by continuously dividing intestinal stem cells (ISCs) resident at the crypt niche. However, the cell type(s) enabling replenishment upon damage and subsequent loss of whole crypts remain largely unclear. Using Set domain-containing protein 4 (Setd4), we identify a small population with reserve stem cell characteristics in the mouse intestine. Upon irradiation-induced injury, Setd4-expressing (Setd4+) cells survive radiation exposure and then activate to produce Sca-1-expressing cell types to restore the epithelial wall and regenerate crypts de novo via crypt fission. Setd4+ cells are confirmed to originate from the early fetal period, subsequently contributing to the development of embryonic gut and the establishment of postnatal crypts. Setd4+ cells are therefore represented as both originators and key regenerators of the intestine.
Cheung, V;Chung, P;Bjorni, M;Shvareva, VA;Lopez, YC;Feinberg, EH;
PMID: 34936877 | DOI: 10.1016/j.celrep.2021.110131
Behavior arises from concerted activity throughout the brain. Consequently, a major focus of modern neuroscience is defining the physiology and behavioral roles of projection neurons linking different brain areas. Single-cell RNA sequencing has facilitated these efforts by revealing molecular determinants of cellular physiology and markers that enable genetically targeted perturbations such as optogenetics, but existing methods for sequencing defined projection populations are low throughput, painstaking, and costly. We developed a straightforward, multiplexed approach, virally encoded connectivity transgenic overlay RNA sequencing (VECTORseq). VECTORseq repurposes commercial retrogradely infecting viruses typically used to express functional transgenes (e.g., recombinases and fluorescent proteins) by treating viral transgene mRNA as barcodes within single-cell datasets. VECTORseq is compatible with different viral families, resolves multiple populations with different projection targets in one sequencing run, and identifies cortical and subcortical excitatory and inhibitory projection populations. Our study provides a roadmap for high-throughput identification of neuronal subtypes based on connectivity.
