scholarly journals scRNA-sequencing reveals subtype-specific transcriptomic perturbations in DRG neurons of Pirt-EGFPf mice in neuropathic pain condition

2022 ◽  
Author(s):  
Chi Zhang ◽  
Ming-Wen Hu ◽  
Shaoqiu He ◽  
Xuewei Wang ◽  
Xu Cao ◽  
...  

Functionally distinct subtypes/clusters of dorsal root ganglion (DRG) neurons, which differ in soma size and neurochemical properties, may play different roles in nerve regeneration and pain. However, details about transcriptomic changes in different neuronal subtypes under maladaptive neuropathic pain conditions remain unclear. Chronic constriction injury (CCI) of the sciatic nerve represents a well-established model of neuropathic pain that mimics the etiology of clinical conditions. Therefore, we conducted single-cell RNA-sequencing (scRNA-seq) to characterize subtype-specific perturbations of transcriptomes in lumbar DRG neurons 7 days after sciatic CCI. By using Pirt-EGFPf mice that selectively express enhanced green fluorescent protein in DRG neurons, we established a highly efficient purification process to enrich neurons for scRNA-seq. We observed a loss of marker genes in injured neurons of 12 standard neuronal clusters, and the emergence of four prominent CCI-induced clusters at this peak-maintenance phase of neuropathic pain. Importantly, a portion of injured neurons from a subset of the 12 standard clusters (NP1, PEP5, NF1, and NF2) were spared from injury-induced identity loss, suggesting subtype-specific transcriptomic changes in injured neurons. Moreover, uninjured neurons, which are necessary for mediating the evoked pain, also demonstrated subtype-specific transcriptomic perturbations in these clusters, but not others. Notably, male and female mice showed differential transcriptomic changes in multiple neuronal clusters after CCI, suggesting transcriptomic sexual dimorphism in primary sensory neurons after nerve injury. Collectively, these findings may contribute to the identification of new target genes and development of DRG neuron subtype-specific therapies for optimizing neuropathic pain treatment and nerve regeneration.

Blood ◽  
2010 ◽  
Vol 116 (3) ◽  
pp. e1-e11 ◽  
Author(s):  
Anna Zakrzewska ◽  
Chao Cui ◽  
Oliver W. Stockhammer ◽  
Erica L. Benard ◽  
Herman P. Spaink ◽  
...  

AbstractThe Spi1/Pu.1 transcription factor plays a crucial role in myeloid cell development in vertebrates. Despite extensive studies of Spi1, the controlled gene group remains largely unknown. To identify genes dependent on Spi1, we used a microarray strategy using a knockdown approach in zebrafish embryos combined with fluorescence-activated cell sorting of myeloid cells from transgenic embryos. This approach of using knockdowns with specific green fluorescent protein-marked cell types was highly successful in identifying macrophage-specific genes in Spi1-directed innate immunity. We found a gene group down-regulated on spi1 knockdown, which is also enriched in fluorescence-activated cell-sorted embryonic myeloid cells of a spi1:GFP transgenic line. This gene group, representing putative myeloid-specific Spi1 target genes, contained all 5 previously identified Spi1-dependent zebrafish genes as well as a large set of novel immune-related genes. Colocalization studies with neutrophil and macrophage markers revealed that genes cxcr3.2, mpeg1, ptpn6, and mfap4 were expressed specifically in early embryonic macrophages. In a functional approach, we demonstrated that gene cxcr3.2, coding for chemokine receptor 3.2, is involved in macrophage migration to the site of bacterial infection. Therefore, based on our combined transcriptome analyses, we discovered novel early macrophage-specific marker genes, including a signal transducer pivotal for macrophage migration in the innate immune response.


2015 ◽  
Vol 21 (13) ◽  
pp. 1723-1740 ◽  
Author(s):  
Katarzyna Panczyk ◽  
Sylwia Go.lda ◽  
Anna Waszkielewicz ◽  
Dorota Zelaszczyk ◽  
Agnieszka Gunia-Krzyz.ak ◽  
...  

2021 ◽  
Vol 9 (8) ◽  
pp. 1570
Author(s):  
Chien-Hsun Huang ◽  
Chih-Chieh Chen ◽  
Yu-Chun Lin ◽  
Chia-Hsuan Chen ◽  
Ai-Yun Lee ◽  
...  

The current taxonomy of the Lactiplantibacillus plantarum group comprises of 17 closely related species that are indistinguishable from each other by using commonly used 16S rRNA gene sequencing. In this study, a whole-genome-based analysis was carried out for exploring the highly distinguished target genes whose interspecific sequence identity is significantly less than those of 16S rRNA or conventional housekeeping genes. In silico analyses of 774 core genes by the cano-wgMLST_BacCompare analytics platform indicated that csbB, morA, murI, mutL, ntpJ, rutB, trmK, ydaF, and yhhX genes were the most promising candidates. Subsequently, the mutL gene was selected, and the discrimination power was further evaluated using Sanger sequencing. Among the type strains, mutL exhibited a clearly superior sequence identity (61.6–85.6%; average: 66.6%) to the 16S rRNA gene (96.7–100%; average: 98.4%) and the conventional phylogenetic marker genes (e.g., dnaJ, dnaK, pheS, recA, and rpoA), respectively, which could be used to separat tested strains into various species clusters. Consequently, species-specific primers were developed for fast and accurate identification of L. pentosus, L. argentoratensis, L. plantarum, and L. paraplantarum. During this study, one strain (BCRC 06B0048, L. pentosus) exhibited not only relatively low mutL sequence identities (97.0%) but also a low digital DNA–DNA hybridization value (78.1%) with the type strain DSM 20314T, signifying that it exhibits potential for reclassification as a novel subspecies. Our data demonstrate that mutL can be a genome-wide target for identifying and classifying the L. plantarum group species and for differentiating novel taxa from known species.


Insects ◽  
2021 ◽  
Vol 12 (1) ◽  
pp. 41
Author(s):  
Ya-Wen Chang ◽  
Yu-Cheng Wang ◽  
Xiao-Xiang Zhang ◽  
Junaid Iqbal ◽  
Yu-Zhou Du

The leafminer fly, Liriomyza trifolii, is an invasive pest of vegetable and horticultural crops in China. In this study, a microinjection method based on dsRNA was developed for RNA interference (RNAi) in L. trifolii using genes encoding vacuolar-ATPase (V-ATPase). Expression analysis indicated that V-ATPase B and V-ATPase D were more highly expressed in L. trifolii adults than in larvae or pupae. Microinjection experiments with dsV-ATPase B and dsV-ATPase D were conducted to evaluate the efficacy of RNAi in L. trifolii adults. Expression analysis indicated that microinjection with 100 ng dsV-ATPase B or dsV-ATPase led to a significant reduction in V-ATPase transcripts as compared to that of the dsGFP control (dsRNA specific to green fluorescent protein). Furthermore, lower dsRNA concentrations were also effective in reducing the expression of target genes when delivered by microinjection. Mortality was significantly higher in dsV-ATPase B- and dsV-ATPase D-treated insects than in controls injected with dsGFP. The successful deployment of RNAi in L. trifolii will facilitate functional analyses of vital genes in this economically-important pest and may ultimately result in new control strategies.


2015 ◽  
Vol 112 (41) ◽  
pp. 12711-12716 ◽  
Author(s):  
Andrea M. Brum ◽  
Jeroen van de Peppel ◽  
Cindy S. van der Leije ◽  
Marijke Schreuders-Koedam ◽  
Marco Eijken ◽  
...  

Osteoporosis is a common skeletal disorder characterized by low bone mass leading to increased bone fragility and fracture susceptibility. In this study, we have identified pathways that stimulate differentiation of bone forming osteoblasts from human mesenchymal stromal cells (hMSCs). Gene expression profiling was performed in hMSCs differentiated toward osteoblasts (at 6 h). Significantly regulated genes were analyzed in silico, and the Connectivity Map (CMap) was used to identify candidate bone stimulatory compounds. The signature of parbendazole matches the expression changes observed for osteogenic hMSCs. Parbendazole stimulates osteoblast differentiation as indicated by increased alkaline phosphatase activity, mineralization, and up-regulation of bone marker genes (alkaline phosphatase/ALPL, osteopontin/SPP1, and bone sialoprotein II/IBSP) in a subset of the hMSC population resistant to the apoptotic effects of parbendazole. These osteogenic effects are independent of glucocorticoids because parbendazole does not up-regulate glucocorticoid receptor (GR) target genes and is not inhibited by the GR antagonist mifepristone. Parbendazole causes profound cytoskeletal changes including degradation of microtubules and increased focal adhesions. Stabilization of microtubules by pretreatment with Taxol inhibits osteoblast differentiation. Parbendazole up-regulates bone morphogenetic protein 2 (BMP-2) gene expression and activity. Cotreatment with the BMP-2 antagonist DMH1 limits, but does not block, parbendazole-induced mineralization. Using the CMap we have identified a previously unidentified lineage-specific, bone anabolic compound, parbendazole, which induces osteogenic differentiation through a combination of cytoskeletal changes and increased BMP-2 activity.


Circulation ◽  
2007 ◽  
Vol 116 (suppl_16) ◽  
Author(s):  
Kensuke Kimura ◽  
Masaki Ieda ◽  
Hideaki Kanazawa ◽  
Takahide Arai ◽  
Takashi Kawakami ◽  
...  

Background : Cardiac hypertrophy induces the fetal isoform of genes (rejuvenation), including contractile proteins, ion channels, and natriuretic peptides. Cardiac sympathetic nerve function is known to be altered in cardiac hypertrophy and congestive heart failure. We recently reported that alteration of cardiac sympathetic nerves (CSN) was caused by their rejuvenation (Circ Res, 2007). The present study was designed to examine the precise characterization of the rejuvenation of CSN in cardiac hypertrophy. Methods and Results : RV hypertrophy was produced by consistent hypoxia (10% O 2 ) in C57/BL6 mice. RV pressure increased to 47 mmHg, and RV/(body weight) ratio increased by 1.6 fold. Nerve growth factor protein was augmented in hypertrophic RV, but was unchanged in LV. Double-transgenic mice, which specifically express eGFP (enhanced green fluorescent protein) in the sympathetic neurons, was generated by crossing dopamine β-hydroxylase (DBH)-Cre mice with Floxed-eGFP mice. The eGFP-positive CSN were markedly increased in hypertrophic RV, but not in LV. Nerve density, quantitated by immunostained area with eGFP and GAP43 (growth-associated corn marker), increased by 8.1 and 9.3 fold, respectively, in RV, but not in LV. (4) Catecholamine content was attenuated in RV. (5) Western blot revealed that tyrosine hydroxylase was markedly down-regulated in RV. (6) Immunostaining clearly demonstrated that the immature neuron markers, PSA-NCAM (highly polysialylated neural cell adhesion molecule) and Ulip-1 (Unc-33-like phosphoprotein 1), were expressed in CSN in hypertrophic RV and stellate ganglia. Basic helix-loop-helix transcription factor, Mash-1 (mammalian achaete-scute complex homolog 1) was strongly expressed in the stellate ganglia. (7) Immature neuron marker-immunopositive cells in stellate ganglia had a markedly decreased TH expression. Conclusion : The rejuvenated CSN showed various immature and fetal neuron marker genes at not only the peripheral axons but also the cellular bodies at the stellate ganglia. Rejuvenation of CSN might be critically involved in the alteration of sympathetic neuronal function in cardiac hypertrophy, including depressed norepinephrine synthesis and hyperinnervation.


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