Array Tomography of zebrafish axillary lymphoid organ - an external, experimentally accessible immune organ in the zebrafish
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ABSTRACT:
SUBMITTER: Kedar Narayan
PROVIDER: EMPIAR-12377 | biostudies-other |
REPOSITORIES: biostudies-other
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Project description:Lymph nodes and other secondary lymphoid organs play critical roles in immune surveillance and immune activation in mammals, but the deep internal locations of these organs makes it challenging to study dynamic cellular mechanisms of immune cell interaction with pathogens or other host tissues such as the vasculature in living animals. Here, we describe a previously uncharacterized external immune organ located near the base of the zebrafish pectoral fin, the pectoral axillary lobe (PAL), with a variety of features that make it ideally suited for studying immune cell dynamics in vivo. This small, transparent organ consists of an outer cortex teeming with immune cells and an inner medulla with a mesh-like network of fibroblastic reticular cells along which immune cells migrate, and a network of lymphatic vessels draining to a large adjacent lymph sac. Noninvasive high-resolution imaging of transgenically marked immune cells can be carried out in the lobes of living animals, and the PAL is readily accessible to external treatment with antigens or pathogens. This newly discovered tissue provides a superb model for dynamic live imaging of immune cells and their interaction with pathogens and surrounding tissues, including blood and lymphatic vessels.
2024-08-26 | GSE270797 | GEO
Project description:The axillary lymphoid organ - an external, experimentally accessible immune organ in the zebrafish
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Project description:For 3D reconstructions of whole immune cells from zebrafish, isolated from adult animals by FAC-sorting we employed array tomography on hundreds of serial sections deposited on silicon wafers. Image stacks were either recorded manually or automatically with the newly released ZEISS Atlas 5 Array Tomography platform on a Zeiss FEGSEM. To characterize different populations of immune cells, organelle inventories were created by segmenting individual cells. In addition, arrays were used for quantification of cell populations with respect to the various cell types they contained. The detection of immunological synapses in cocultures of cell populations from thymus or WKM with cancer cells helped to identify the cytotoxic nature of these cells. Our results demonstrate the practicality and benefit of AT for high-throughput ultrastructural imaging of substantial volumes.
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Project description:Bovine Viral Diarrhea Virus (BVDV) fetal infections occur in two forms; persistent infection (PI) or transient infection (TI), depending on what stage of gestation the fetus is infected. Examination of lymphoid organs from both PI and TI fetuses reveals drastically different fetal responses, dependent upon the developmental stage of the fetal immune system. Total RNA was extracted from the thymuses and spleens of uninfected control, PI, and TI fetuses collected on day 190 of gestation to test the hypothesis that BVDV infection impairs the innate and adaptive immune response in the fetal thymus and spleen of both infection types. Transcripts of genes representing the innate immune response and adaptive immune response genes were assayed by Reverse Transcription quatitative PCR (RT-qPCR) (2-??Cq; fold change). Genes of the innate immune response, interferon (IFN) inducible genes, antigen presentation to lymphocytes, and activation of B cells were downregulated in day 190 fetal PI thymuses compared to controls. In contrast, innate immune response genes were upregulated in TI fetal thymuses compared to controls and tended to be upregulated in TI fetal spleens. Genes associated with the innate immune system were not different in PI fetal spleens; however, adaptive immune system genes were downregulated, indicating that PI fetal BVDV infection has profound inhibitory effects on the expression of genes involved in the innate and adaptive immune response. The downregulation of these genes in lymphocytes and antigen-presenting cells in the developing thymus and spleen may explain the incomplete clearance of BVDV and the persistence of the virus in PI animals while the upregulation of the TI innate immune response indicates a more mature immune system, able to clear the virus.
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Project description:Chromosomal translocations are a hallmark of hematopoietic malignancies. CG motifs within translocation fragile zones (typically 20 to 600 bp in size) are prone to chromosomal translocation in lymphomas. Here we demonstrate that the CG motifs in human translocation fragile zones are hypomethylated relative to the adjacent DNA. Using a methyltransferase footprinting assay on isolated nuclei (in vitro), we find that the chromatin at these fragile zones is accessible. We also examined in vivo accessibility using cellular expression of a prokaryotic methylase. Based on this assay, which measures accessibility over a much longer time interval than is possible with in vitro methods, these fragile zones were found to be more accessible than the adjacent DNA. Because DNA within the fragile zones can be methylated by both cellular and exogenous methyltransferases, the fragile zones are predominantly in a duplex DNA conformation. These observations permit more-refined models for why these zones are 100- to 1,000-fold more prone to undergo chromosomal translocation than the adjacent regions.
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Project description:BackgroundRecently, a physical model of nucleosome formation based on sequence-dependent bending properties of the DNA double-helix has been used to reveal some enrichment of nucleosome-inhibiting energy barriers (NIEBs) nearby ubiquitous human "master" replication origins. Here we use this model to predict the existence of about 1.6 millions NIEBs over the 22 human autosomes.ResultsWe show that these high energy barriers of mean size 153 bp correspond to nucleosome-depleted regions (NDRs) in vitro, as expected, but also in vivo. On either side of these NIEBs, we observe, in vivo and in vitro, a similar compacted nucleosome ordering, suggesting an absence of chromatin remodeling. This nucleosomal ordering strongly correlates with oscillations of the GC content as well as with the interspecies and intraspecies mutation profiles along these regions. Comparison of these divergence rates reveals the existence of both positive and negative selections linked to nucleosome positioning around these intrinsic NDRs. Overall, these NIEBs and neighboring nucleosomes cover 37.5 % of the human genome where nucleosome occupancy is stably encoded in the DNA sequence. These 1 kb-sized regions of intrinsic nucleosome positioning are equally found in GC-rich and GC-poor isochores, in early and late replicating regions, in intergenic and genic regions but not at gene promoters.ConclusionThe source of selection pressure on the NIEBs has yet to be resolved in future work. One possible scenario is that these widely distributed chromatin patterns have been selected in human to impair the condensation of the nucleosomal array into the 30 nm chromatin fiber, so as to facilitate the epigenetic regulation of nuclear functions in a cell-type-specific manner.
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Project description:Advancing our understanding of embryonic development is heavily dependent on identification of novel pathways or regulators. While genome-wide techniques such as RNA sequencing are ideally suited for discovering novel candidate genes, they are unable to yield spatially resolved information in embryos or tissues. Microscopy-based approaches, using for example in situ hybridization, can provide spatial information about gene expression, but are limited to analyzing one or a few genes at a time. Here, we present a method where we combine traditional histological techniques with low-input RNA sequencing and mathematical image reconstruction to generate a high-resolution genome-wide 3D atlas of gene expression in the zebrafish embryo at three developmental stages. We also demonstrate that our technique is suitable for spatially-resolved differential expression analysis in wildtype and Gli3 mutant mouse forelimbs. Importantly, our method enables searching for genes that are expressed in specific spatial patterns without manual image annotation. We envision broad applicability of RNA tomography as an accurate and sensitive approach for spatially resolved transcriptomics in whole embryos and dissected organs.
2014-10-23 | GSE59873 | GEO
Project description:Background and purposeOrgan motion is a challenge during high-precision external beam radiotherapy in cervical cancer, and improved strategies for treatment adaptation and monitoring of target dose coverage are needed. This study evaluates a cone beam computed tomography (CBCT)-based approach.Materials and methodsIn twenty-three patients, individualized internal target volumes (ITVs) were generated from pre-treatment MRI and CT scans with full and empty bladders. The target volumes encompassed high-risk clinical target volume (CTV-T HR) (gross tumor volume + remaining cervix) and low risk (LR) CTV-T (CTV-T HR + uterus + parametriae + upper vagina). Volumetric Modulated Arc Therapy (VMAT) was used to deliver a dose of 45 Gy in 25 fractions. CBCTs were used for setup and for radiation therapists (RTTs) to evaluate the target coverage (inside/outside the planning target volume). CBCTs were reviewed offline. Estimates of the dose delivered with minimum (point) doses across all fractions to CTV-T HR (aim 42.75 Gy) and CTV-T LR (aim 40 Gy) were assessed. In patients with insufficient dose coverage, re-plans were generated based on previous imaging.ResultsMedian (range) of the ITV-margins (mean of anterior-posterior margins) related to uterus and cervix was 1.2 (0.5-2.2 and 1.0-2.1) cm. RTTs were able to assess the target coverage in 90% of all CBCTs (505/563). With re-planning, one patient had considerable benefit (12.7 Gy increase of minimum dose) to CTV-T LR_vagina, four patients had improved dose to the CTV-T LR_uterus (1.2-1.8 Gy), and 3 patients did not benefit from re-planning.ConclusionsDaily CBCT-based monitoring of target coverage by the RTTs has proven safe with limited workload. It allows for reduction in the treated volumes without compromising the target dose coverage.
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Project description:The lymphoid organ is an essential part of the immune system involved in cellular and humoral immune responses in shrimp. However, its roles in the immune responses against different pathogens are still largely unclear. In the present study, transcriptomic analysis was applied to compare the differentially expressed genes (DEGs) in the lymphoid organ of shrimp after Vibrio or WSSV challenge. In total, 2127 DEGs were screened in the lymphoid organ of shrimp at 6 h post Vibrio parahaemolyticus injection, and 1569 DEGs were obtained at the same time after WSSV challenge. KEGG pathway enrichment analysis of these DEGs revealed that two significantly enriched pathways including "neuroactive ligand-receptor interaction" and "protein digestion and absorption" were responsive to both pathogens. In contrast, "lysosome" was the significantly enriched pathway only in Vibrio challenge whereas carbohydrate metabolism related pathways were the significantly enriched pathways only in WSSV challenge. Further analysis on immune-related DEGs showed that Vibrio challenge induced broad immune responses in the lymphoid organ including activation of several pattern recognition receptors, the proPO activating system, phagocytosis related genes, and immune effectors. In contrast, the immune responses seemed to be inhibited after WSSV infection. The data suggest that the shrimp lymphoid organ plays different functions in response to the infection of distinct pathogens at the early stage, which provides new insights into the immune functions of lymphoid organ in shrimp.
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Project description:Advancing our understanding of embryonic development is heavily dependent on identification of novel pathways or regulators. While genome-wide techniques such as RNA sequencing are ideally suited for discovering novel candidate genes, they are unable to yield spatially resolved information in embryos or tissues. Microscopy-based approaches, using for example in situ hybridization, can provide spatial information about gene expression, but are limited to analyzing one or a few genes at a time. Here, we present a method where we combine traditional histological techniques with low-input RNA sequencing and mathematical image reconstruction to generate a high-resolution genome-wide 3D atlas of gene expression in the zebrafish embryo at three developmental stages. We also demonstrate that our technique is suitable for spatially-resolved differential expression analysis in wildtype and Gli3 mutant mouse forelimbs. Importantly, our method enables searching for genes that are expressed in specific spatial patterns without manual image annotation. We envision broad applicability of RNA tomography as an accurate and sensitive approach for spatially resolved transcriptomics in whole embryos and dissected organs. To generate spatially-resolved RNA-seq data for zebrafish embryos (shield stage, 10 somites, 15 somites, 18 somites) and mouse forelimbs (E10.5), we cryosectioned samples, extracted RNA from the individual sections, and amplified and barcoded mRNA using the CEL-seq protocol (Hashimshony et al., Cell Reports, 2012) with a few modifications. Libraries were sequenced on Illumina HiSeq 2500 using 50bp paired end sequencing. Selected zebrafish libraries were sequenced on MiSeq 250bp paired-end to improve 3' annotations.
2014-10-23 | E-GEOD-59873 | biostudies-arrayexpress