scholarly journals scRNA Transcription Profile of Adult Zebrafish Podocytes Using a Novel Reporter Strain

2021 ◽  
Vol 55 (S4) ◽  
pp. 35-47
Keyword(s):  
Calcium Binding ◽  
Kidney Development ◽  
Light Sheet ◽  
Transgenic Zebrafish ◽  
Fluorescent Reporter ◽  
Single Plane ◽  
Fluorescent Reporters ◽  
Injury And Repair ◽  
Gene Switching

BACKGROUND/AIMS: The role of podocytes is well conserved across species from drosophila to teleosts, and mammals. Identifying the molecular markers that actively maintain the integrity of the podocyte will enable a greater understanding of the changes that lead to damage. METHODS: We generated transgenic zebrafish, expressing fluorescent reporters driven by the podocin promoter, for the visualization and isolation of podocytes. We have conducted single cell RNA sequencing (scRNA-seq) on isolated podocytes from a zebrafish reporter line. RESULTS: We demonstrated that the LifeAct-TagRFP-T fluorescent reporter faithfully replicated podocin expression in vivo. We were also able to show spontaneous GCaMP6s fluorescence using light sheet (single plane illumination) microscopy. We identified many podocyte transcripts, encoding proteins related to calcium-binding and actin filament assembly, in common with those expressed in human and mouse mature podocytes. CONCLUSION: We describe the establishment of novel transgenic zebrafish and their use to identify and isolate podocyte cells for the preparation of a scRNA-seq library from normal podocytes. The scRNA-seq data identifies distinct populations of cells and potential gene switching between clusters. These data provide a foundation for future comparative studies and for exploiting the zebrafish as a model for kidney development, disease, injury and repair.

scholarly journals Enhancement and Segmentation Workflow for the Developing Zebrafish Vasculature

2019 ◽  
Vol 5 (1) ◽  
pp. 14 ◽  
Author(s):  
Elisabeth Kugler ◽  
Karen Plant ◽  
Timothy Chico ◽  
Paul Armitage
Keyword(s):  
Open Source Software ◽  
Light Sheet ◽  
Visual Assessment ◽  
Cardiovascular Development ◽  
Fluorescent Reporter ◽  
Enhancement Method ◽  
Vertebrate Model ◽  
Vascular Enhancement ◽  
Light Sheet Fluorescence Microscopy

Zebrafish have become an established in vivo vertebrate model to study cardiovascular development and disease. However, most published studies of the zebrafish vascular architecture rely on subjective visual assessment, rather than objective quantification. In this paper, we used state-of-the-art light sheet fluorescence microscopy to visualize the vasculature in transgenic fluorescent reporter zebrafish. Analysis of image quality, vascular enhancement methods, and segmentation approaches were performed in the framework of the open-source software Fiji to allow dissemination and reproducibility. Here, we build on a previously developed image processing pipeline; evaluate its applicability to a wider range of data; apply and evaluate an alternative vascular enhancement method; and, finally, suggest a work-flow for successful segmentation of the embryonic zebrafish vasculature.

scholarly journals In vivo imaging of zebrafish digestive organ function using multiple quenched fluorescent reporters

2009 ◽  
Vol 296 (2) ◽  
pp. G445-G453 ◽  
Author(s):  
Kotaro Hama ◽  
Elayne Provost ◽  
Timothy C. Baranowski ◽  
Amy L. Rubinstein ◽  
Jennifer L. Anderson ◽  
...  
Keyword(s):  
Protease Activity ◽  
Fluorescent Protein ◽  
Digestive Organ ◽  
Phospholipase Activity ◽  
Organ Function ◽  
Fluorescent Reporter ◽  
Fluorescent Reporters ◽  
Zebrafish Larvae ◽  
Intestinal Protein

Optical clarity of larvae makes the zebrafish ideal for real-time analyses of vertebrate organ function through the use of fluorescent reporters of enzymatic activities. A key function of digestive organs is to couple the generation of enzymes with mechanical processes that enable nutrient availability and absorption. However, it has been extremely difficult, and in many cases not possible, to directly observe digestive processes in a live vertebrate. Here we describe a new method to visualize intestinal protein and lipid processing simultaneously in live zebrafish larvae using a quenched fluorescent protein (EnzChek) and phospholipid (PED6). By employing these reagents, we found that wild-type larvae exhibit significant variation in intestinal phospholipase and protease activities within a group but display a strong correlation between the activities within individuals. Furthermore, we found that pancreas function is essential for larval digestive protease activity but not for larval intestinal phospholipase activity. Although fat-free ( ffr) mutant larvae were previously described to exhibit impaired lipid processes, we found they also had significantly reduced protease activity. Finally, we selected and evaluated compounds that were previously suggested to have altered phospholipase activity and are known or suspected to have inflammatory effects in the intestinal tract including nonsteroidal anti-inflammatory drugs, and identified a compound that significantly increases intestinal phospholipid processing. Thus the multiple fluorescent reporter-based methodology facilitates the rapid analysis of digestive organ function in live zebrafish larvae.

scholarly journals Development of High-Content Assays for Kidney Progenitor Cell Expansion in Transgenic Zebrafish

2013 ◽  
Vol 18 (10) ◽  
pp. 1193-1202 ◽  
Author(s):  
Subramaniam Sanker ◽  
Maria Cecilia Cirio ◽  
Laura L. Vollmer ◽  
Natasha D. Goldberg ◽  
Lee A. McDermott ◽  
...  
Keyword(s):  
Artificial Intelligence ◽  
Image Analysis ◽  
Time Course ◽  
Kidney Development ◽  
Egfp Expression ◽  
Transgenic Zebrafish ◽  
Zebrafish Embryos ◽  
Kidney Regeneration ◽  
Kidney Organogenesis

Reactivation of genes normally expressed during organogenesis is a characteristic of kidney regeneration. Enhancing this reactivation could potentially be a therapeutic target to augment kidney regeneration. The inductive events that drive kidney organogenesis in zebrafish are similar to the initial steps in mammalian kidney organogenesis. Therefore, quantifying embryonic signals that drive zebrafish kidney development is an attractive strategy for the discovery of potential novel therapeutic modalities that accelerate kidney regeneration. The Lim1 homeobox protein, Lhx1, is a marker of kidney development that is also expressed in the regenerating kidneys after injury. Using a fluorescent Lhx1a-EGFP transgene whose phenotype faithfully recapitulates that of the endogenous protein, we developed a high-content assay for Lhx1a-EGFP expression in transgenic zebrafish embryos employing an artificial intelligence–based image analysis method termed cognition network technology (CNT). Implementation of the CNT assay on high-content readers enabled automated real-time in vivo time-course, dose-response, and variability studies in the developing embryo. The Lhx1a assay was complemented with a kidney-specific secondary CNT assay that enables direct measurements of the embryonic renal tubule cell population. The integration of fluorescent transgenic zebrafish embryos with automated imaging and artificial intelligence–based image analysis provides an in vivo analysis system for structure-activity relationship studies and de novo discovery of novel agents that augment innate regenerative processes.

scholarly journals Identification and Characterization of a Novel Protein ASP-3 Purified from Arca subcrenata and Its Antitumor Mechanism

Marine Drugs ◽  
2019 ◽  
Vol 17 (9) ◽  
pp. 528 ◽  
Author(s):  
Zhongyi Guo ◽  
Hui Shi ◽  
Chunlei Li ◽  
Yuanyuan Luo ◽  
Sixue Bi ◽  
...  
Keyword(s):  
Calcium Binding ◽  
Hepg2 Cells ◽  
Therapeutic Potential ◽  
Umbilical Vein ◽  
In Vivo Model ◽  
Transgenic Zebrafish ◽  
Zebrafish Model ◽  
Novel Protein

Diverse bioactive substances derived from marine organisms have been attracting growing attention. Besides small molecules and polypeptides, numerous studies have shown that marine proteins also exhibit antitumor activities. Small anticancer proteins can be expressed in vivo by viral vectors to exert local and long-term anticancer effects. Herein, we purified and characterized a novel protein (ASP-3) with unique antitumor activity from Arca subcrenata Lischke. The ASP-3 contains 179 amino acids with a molecular weight of 20.6 kDa. The spectral characterization of ASP-3 was elucidated using Fourier Transform infrared spectroscopy (FTIR) and Circular Dichroism (CD) spectroscopy. Being identified as a sarcoplasmic calcium-binding protein, ASP-3 exhibited strong inhibitory effects on the proliferation of Human hepatocellular carcinoma (HepG2) cells with an IC50 value of 171.18 ± 18.59 μg/mL, measured by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. The RNA-seq analysis showed that ASP-3 regulated the vascular endothelial growth factor receptor (VEGFR) signaling pathway in HepG2 cells. Immunofluorescence results indicated that ASP-3 effectively reduced VEGFR2 phosphorylation in HepG2 cells and affected the downstream components of VEGF signaling pathways. The surface plasmon resonance (SPR) analysis further demonstrated that ASP-3 direct interacted with VEGFR2. More importantly, the therapeutic potential of ASP-3 as an anti-angiogenesis agent was further confirmed by an in vitro model using VEGF-induced tube formation assay of human umbilical vein endothelial cells (HUVECs), as well as an in vivo model using transgenic zebrafish model. Taken together, the ASP-3 provides a good framework for the development of even more potent anticancer proteins and provides important weapon for cancer treatment using novel approaches such as gene therapy.

scholarly journals CRISPR/Cas9 mediated dual fluorescent reporter mice to study spatial bone genomics of osteoblasts and osteoclasts in the local in vivo environment

Bone Reports ◽  
2021 ◽  
Vol 14 ◽  
pp. 100871
Author(s):  
Dilara Yilmaz ◽  
Yannick Fischer ◽  
Sandra Zimmermann ◽  
Gaonhae Hwang ◽  
Ralph Müller ◽  
...  
Keyword(s):  
Fluorescent Reporter ◽  
Reporter Mice

scholarly journals Imaging the mechanisms of anti-CD20 therapy in vivo uncovers spatiotemporal bottlenecks in antibody-dependent phagocytosis

2021 ◽  
Vol 7 (8) ◽  
pp. eabd6167
Author(s):  
Capucine L. Grandjean ◽  
Zacarias Garcia ◽  
Fabrice Lemaître ◽  
Béatrice Bréart ◽  
Philippe Bousso
Keyword(s):  
B Cell ◽  
Cell Lymphoma ◽  
Critical Path ◽  
B Cell Lymphoma ◽  
Antibody Dependent Cellular Cytotoxicity ◽  
Fluorescent Reporter ◽  
Cd20 Antibody ◽  
Anti Cd20 ◽  
Partial Depletion

Anti-CD20 antibody (mAb) represents an effective strategy for the treatment of B cell malignancies, possibly involving complement activity, antibody-dependent cellular cytotoxicity and phagocytosis (ADP). While ADP by Kupffer cells deplete circulating tumors, mechanisms targeting non-circulating tumors remain unclear. Using intravital imaging in a model of B cell lymphoma, we establish here the dominance and limitations of ADP in the bone marrow (BM). We found that tumor cells were stably residing in the BM with little evidence for recirculation. To elucidate the mechanism of depletion, we designed a dual fluorescent reporter to visualize phagocytosis and apoptosis. ADP by BM-associated macrophages was the primary mode of tumor elimination but was no longer active after one hour, resulting in partial depletion. Moreover, macrophages were present at low density in tumor-rich regions, targeting only neighboring tumors. Overcoming spatiotemporal bottlenecks in tumor-targeting Ab therapy thus represents a critical path towards the design of optimized therapies.

scholarly journals In-vivo 3D imaging of Zebrafish’s intersegmental vessel development by a bi-directional light-sheet illumination microscope

2021 ◽  
Vol 557 ◽  
pp. 8-13
Author(s):  
Xiaofei Qin ◽  
Chong Chen ◽  
Linbo Wang ◽  
Xiaohu Chen ◽  
Yong Liang ◽  
...  
Keyword(s):  
3D Imaging ◽  
Light Sheet ◽  
Vessel Development

scholarly journals Development of BCR-ABL1 Transgenic Zebrafish Model Reproducing Chronic Myeloid Leukemia (CML) Like-Disease and Providing a New Insight into CML Mechanisms

Cells ◽  
2021 ◽  
Vol 10 (2) ◽  
pp. 445
Author(s):  
Daniela Zizioli ◽  
Simona Bernardi ◽  
Marco Varinelli ◽  
Mirko Farina ◽  
Luca Mignani ◽  
...  
Keyword(s):  
Chronic Myeloid Leukemia ◽  
Myeloid Leukemia ◽  
Fusion Gene ◽  
Philadelphia Chromosome ◽  
Leukemic Cells ◽  
Transgenic Zebrafish ◽  
Hematopoietic Tissue ◽  
Zebrafish Model ◽  
Altered Expression

Zebrafish has proven to be a versatile and reliable experimental in vivo tool to study human hematopoiesis and model hematological malignancies. Transgenic technologies enable the generation of specific leukemia types by the expression of human oncogenes under specific promoters. Using this technology, a variety of myeloid and lymphoid malignancies zebrafish models have been described. Chronic myeloid leukemia (CML) is a clonal myeloproliferative neoplasia characterized by the BCR-ABL1 fusion gene, derived from the t (9;22) translocation causing the Philadelphia Chromosome (Ph). The BCR-ABL1 protein is a constitutively activated tyrosine kinas inducing the leukemogenesis and resulting in an accumulation of immature leukemic cells into bone marrow and peripheral blood. To model Ph+ CML, a transgenic zebrafish line expressing the human BCR-ABL1 was generated by the Gal4/UAS system, and then crossed with the hsp70-Gal4 transgenic line. The new line named (BCR-ABL1pUAS:CFP/hsp70-Gal4), presented altered expression of hematopoietic markers during embryonic development compared to controls and transgenic larvae showed proliferating hematopoietic cells in the caudal hematopoietic tissue (CHT). The present transgenic zebrafish would be a robust CML model and a high-throughput drug screening tool.

scholarly journals Exosomal S100A4 derived from highly metastatic hepatocellular carcinoma cells promotes metastasis by activating STAT3

2021 ◽  
Vol 6 (1) ◽  
Author(s):  
Haoting Sun ◽  
Chaoqun Wang ◽  
Beiyuan Hu ◽  
Xiaomei Gao ◽  
Tiantian Zou ◽  
...  
Keyword(s):  
Hepatocellular Carcinoma ◽  
Cancer Progression ◽  
Calcium Binding ◽  
Tumor Metastasis ◽  
Metastatic Potential ◽  
Functional Roles ◽  
Stat3 Phosphorylation ◽  
Hcc Cells

AbstractIntercellular cross-talk plays important roles in cancer progression and metastasis. Yet how these cancer cells interact with each other is still largely unknown. Exosomes released by tumor cells have been proved to be effective cell-to-cell signal mediators. We explored the functional roles of exosomes in metastasis and the potential prognostic values for hepatocellular carcinoma (HCC). Exosomes were extracted from HCC cells of different metastatic potentials. The metastatic effects of exosomes derived from highly metastatic HCC cells (HMH) were evaluated both in vitro and in vivo. Exosomal proteins were identified with iTRAQ mass spectrum and verified in cell lines, xenograft tumor samples, and functional analyses. Exosomes released by HMH significantly enhanced the in vitro invasion and in vivo metastasis of low metastatic HCC cells (LMH). S100 calcium-binding protein A4 (S100A4) was identified as a functional factor in exosomes derived from HMH. S100A4rich exosomes significantly promoted tumor metastasis both in vitro and in vivo compared with S100A4low exosomes or controls. Moreover, exosomal S100A4 could induce expression of osteopontin (OPN), along with other tumor metastasis/stemness-related genes. Exosomal S100A4 activated OPN transcription via STAT3 phosphorylation. HCC patients with high exosomal S100A4 in plasma also had a poorer prognosis. In conclusion, exosomes from HMH could promote the metastatic potential of LMH, and exosomal S100A4 is a key enhancer for HCC metastasis, activating STAT3 phosphorylation and up-regulating OPN expression. This suggested exosomal S100A4 to be a novel prognostic marker and therapeutic target for HCC metastasis.

scholarly journals Neuroprotective Effect of Optimized Yinxieling Formula in 6-OHDA-Induced Chronic Model of Parkinson’s Disease through the Inflammation Pathway

2019 ◽  
Vol 2019 ◽  
pp. 1-11 ◽  
Author(s):  
Renrong Wei ◽  
Cuiping Rong ◽  
Qingfeng Xie ◽  
Shouhai Wu ◽  
Yuchao Feng ◽  
...  
Keyword(s):  
Parkinson’S Disease ◽  
Parkinson's Disease ◽  
Calcium Binding ◽  
Dopaminergic Neurons ◽  
Neuroprotective Effect ◽  
Interleukin 1 ◽  
Mrna Levels ◽  
Cox 2

Parkinson’s disease (PD) is characterized by progressive degeneration of dopaminergic neurons in the substantia nigra (SN)-striatum circuit, which is associated with glial activation and consequent chronic neuroinflammation. Optimized Yinxieling Formula (OYF) is a Chinese medicine that exerts therapeutical effect and antiinflammation property on psoriasis. Our previous study has proven that pretreatment with OYF could regulate glia-mediated inflammation in an acute mouse model of PD induced by 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine. Given that PD is a chronic degeneration disorder, this study applied another PD animal model induced by striatal injection of 6-hydroxydopamine (6-OHDA) to mimic the progressive damage of the SN-striatum dopamine system in rats. The OYF was administrated in the manner of pretreatment plus treatment. The effects of the OYF on motor behaviors were assessed with the apomorphine-induced rotation test and adjusting steps test. To confirm the effect of OYF on dopaminergic neurons and glia activation in this model, we analyzed the expression of tyrosine hydroxylase (TH) and glia markers, ionized calcium-binding adapter molecule 1 (Iba-1), and glial fibrillary acidic protein (GFAP) in the SN region of the rat PD model. Inflammation-associated factors, including tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), IL-6, inducible nitric oxide synthase (iNOS), and cyclooxygenase-2 (COX-2), were further evaluated in this model and in interferon-γ- (INF-γ-) induced murine macrophages RAW264.7 cells. The results from the in vivo study showed that OYF reversed the motor behavioral dysfunction in 6-OHDA-induced PD rats, upregulated the TH expression, decreased the immunoreactivity of Iba-1 and GFAP, and downregulated the mRNA levels of TNF-α and COX-2. The OYF also trended to decrease the mRNA levels of IL-1β and iNOS in vivo. The results from the in vitro study showed that OYF significantly decreased the mRNA levels of TNF-α, IL-1β, IL-6, iNOS, and COX-2. Therefore, this study suggests that OYF exerts antiinflammatory effects, which might be related to the protection of dopaminergic neurons in 6-OHDA-induced chronic neurotoxicity.

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