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.
Zwaans BMM, Wegner KA, Bartolone SN, Vezina CM, Chancellor MB, Lamb LE
PMID: 32109348 | DOI: 10.14814/phy2.14377
A subset of patients receiving radiation therapy for pelvic cancer develop radiation cystitis, a complication characterized by mucosal cell death, inflammation, hematuria, and bladder fibrosis. Radiation cystitis can reduce bladder capacity, cause incontinence, and impair voiding function so severely that patients require surgical intervention. Factors influencing onset and severity of radiation cystitis are not fully known. We tested the hypothesis that genetic background is a contributing factor. We irradiated bladders of female C57BL/6, C3H, and BALB/c mice and evaluated urinary voiding function, bladder shape, histology, collagen composition, and distribution of collagen-producing cells. We found that the genetic background profoundly affects the severity of radiation-induced bladder fibrosis and urinary voiding dysfunction. C57BL/6 mice are most susceptible and C3H mice are most resistant. Irradiated C57BL/6 mouse bladders are misshapen and express more abundant collagen I and III proteins than irradiated C3H and BALB/c bladders. We localized Col1a1 and Col3a1 mRNAs to FSP1-negative stromal cells in the bladder lamina propria and detrusor. The number of collagen I and collagen III-producing cells can predict the average voided volume of a mouse. Collectively, we show that genetic factors confer sensitivity to radiation cystitis, establish C57BL/6 mice as a sensitive preclinical model, and identify a potential role for FSP1-negative stromal cells in radiation-induced bladder fibrosis
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.
Gupta K, Levinsohn J, Linderman G, Chen D, Sun TY, Dong D, Taketo MM, Bosenberg M, Kluger Y, Choate K, Myung P.
PMID: 30595533 | DOI: 10.1016/j.devcel.2018.11.032
Delineating molecular and cellular events that precede appendage morphogenesis has been challenging due to the inability to distinguish quantitative molecular differences between cells that lack histological distinction. The hair follicle (HF) dermal condensate (DC) is a cluster of cells critical for HF development and regeneration. Events that presage emergence of this distinctive population are poorly understood. Using unbiased single-cell RNA sequencing and in vivo methods, we infer a sequence of transcriptional states through which DC cells pass that begins prior to HF morphogenesis. Our data indicate that Wnt/β-catenin signaling is required to progress into an intermediate stage that precedes quiescence and differentiation. Further, we provide evidence that quiescent DC cells are recent progeny of selectively proliferating cells present prior to morphogenesis and that are later identified in the peri-DC zone during DC expansion. Together, these findings provide an inferred path of molecular states that lead to DC cell differentiation.
Nature biomedical engineering
You, Y;Tian, Y;Yang, Z;Shi, J;Kwak, KJ;Tong, Y;Estania, AP;Cao, J;Hsu, WH;Liu, Y;Chiang, CL;Schrank, BR;Huntoon, K;Lee, D;Li, Z;Zhao, Y;Zhang, H;Gallup, TD;Ha, J;Dong, S;Li, X;Wang, Y;Lu, WJ;Bahrani, E;Lee, LJ;Teng, L;Jiang, W;Lan, F;Kim, BYS;Lee, AS;
PMID: 36635419 | DOI: 10.1038/s41551-022-00989-w
The success of messenger RNA therapeutics largely depends on the availability of delivery systems that enable the safe, effective and stable translation of genetic material into functional proteins. Here we show that extracellular vesicles (EVs) produced via cellular nanoporation from human dermal fibroblasts, and encapsulating mRNA encoding for extracellular-matrix α1 type-I collagen (COL1A1) induced the formation of collagen-protein grafts and reduced wrinkle formation in the collagen-depleted dermal tissue of mice with photoaged skin. We also show that the intradermal delivery of the mRNA-loaded EVs via a microneedle array led to the prolonged and more uniform synthesis and replacement of collagen in the dermis of the animals. The intradermal delivery of EV-based COL1A1 mRNA may make for an effective protein-replacement therapy for the treatment of photoaged skin.
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.
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.
Foster BL, Ao M, Salmon CR, Chavez MB, Kolli TN, Tran AB, Chu EY, Kantovitz KR, Yadav M, Narisawa S, Millán JL, Nociti Jr FH, Somerman MJ.
PMID: - | DOI: 10.1016/j.bone.2017.12.004
The periodontal complex is essential for tooth attachment and function and includes the mineralized tissues, cementum and alveolar bone, separated by the unmineralized periodontal ligament (PDL). To gain insights into factors regulating cementum-PDL and bone-PDL borders and protecting against ectopic calcification within the PDL, we employed a proteomic approach to analyze PDL tissue from progressive ankylosis knock-out (Ank−/−) mice, featuring reduced PPi, rapid cementogenesis, and excessive acellular cementum. Using this approach, we identified the matrix protein osteopontin (Spp1/OPN) as an elevated factor of interest in Ank−/− mouse molar PDL. We studied the role of OPN in dental and periodontal development and function. During tooth development in wild-type (WT) mice, Spp1 mRNA was transiently expressed by cementoblasts and strongly by alveolar bone osteoblasts. Developmental analysis from 14 to 240 days postnatal (dpn) indicated normal histological structures in Spp1−/− comparable to WT control mice. Microcomputed tomography (micro-CT) analysis at 30 and 90 dpn revealed significantly increased volumes and tissue mineral densities of Spp1−/− mouse dentin and alveolar bone, while pulp and PDL volumes were decreased and tissue densities were increased. However, acellular cementum growth was unaltered in Spp1−/− mice. Quantitative PCR of periodontal-derived mRNA failed to identify potential local compensators influencing cementum in Spp1−/− vs. WT mice at 26 dpn. We genetically deleted Spp1 on the Ank−/− mouse background to determine whether increased Spp1/OPN was regulating periodontal tissues when the PDL space is challenged by hypercementosis in Ank−/− mice. Ank−/−; Spp1−/−double deficient mice did not exhibit greater hypercementosis than that in Ank−/− mice. Based on these data, we conclude that OPN has a non-redundant role regulating formation and mineralization of dentin and bone, influences tissue properties of PDL and pulp, but does not control acellular cementum apposition. These findings may inform therapies targeted at controlling soft tissue calcification.
Christiansen, P;Andreasen, C;Laursen, K;Delaisse, J;Andersen, T;
| DOI: 10.2139/ssrn.4224428
Background: Recruitment and proliferation of osteoprogenitors during the reversal-resorption phase, and their differentiation into mature bone-forming osteoblasts is crucial for initiation of bone formation during bone remodeling. This study investigates the osteoprogenitors’ gradual recruitment, proliferation, and differentiation into bone-forming osteoblasts within intracortical remodeling events of healthy adolescent humans. Methods: The study was conducted on cortical bone specimens from 11 healthy adolescent humans. The osteoprogenitor recruitment route and differentiation into osteoblasts were backtracked using immunostainings and in situ hybridizations with osteoblastic markers (CD271, osterix, collage type 1 and 3). The osteoblastic cell populations were defined based on the pore surfaces and their proliferation index (Ki67), density, and number/circumference were estimated in multiplex-immunofluorescence (Ki67, TRAcP, CD34, SMA) stained sections. Results: During the reversal-resorption phase, osteoclasts are intermixed with osteoblastic reversal cells (COL3A1 high CD271 high COL1A1 low Osterix neg ), which are considered to be spatiotemporal osteoprogenitors of bone-forming osteoblasts. Initiation of bone formation requires a critical density of these osteoblastic reversal cells (43±9 cells/mm), which is reached though proliferation (4.4±0.5% proliferative) and even more so through recruitment of osteoprogenitors, but challenged by the ongoing expansion of the canal circumference. These osteoprogenitors most likely originate from osteoblastic bone lining cells and mainly osteoblastic lumen cells, which expand their population though proliferation (4.6±0.3%) and vascular recruitment. These lumen cells resemble canopy cells above trabecular remodeling sites, and like canopy cells they extend above bone-forming osteoblasts where they may rejuvenate the osteoblast population during bone formation. Conclusion: Initiation of bone formation during intracortical remodeling requires a critical density osteoblastic reversal cells, which is reached though proliferation and recruitment of local osteoprogenitors: bone lining cells and osteoblastic lumen cells.
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.
