scholarly journals Loss of Wnt16 Leads to Skeletal Deformities and Downregulation of Bone Developmental Pathway in Zebrafish

2021 ◽  
Vol 22 (13) ◽  
pp. 6673
Author(s):  
Xiaochao Qu ◽  
Mei Liao ◽  
Weiwei Liu ◽  
Yisheng Cai ◽  
Qiaorong Yi ◽  
...  
Keyword(s):  
Gene Knockout ◽  
Integration Site ◽  
Skeletal Development ◽  
Rna Seq ◽  
Developmental Pathway ◽  
Mineral Density ◽  
Zebrafish Model ◽  
Protein Protein Interaction ◽  
Ct Analysis

Wingless-type MMTV integration site family, member 16 (wnt16), is a wnt ligand that participates in the regulation of vertebrate skeletal development. Studies have shown that wnt16 can regulate bone metabolism, but its molecular mechanism remains largely undefined. We obtained the wnt16-/- zebrafish model using the CRISPR-Cas9-mediated gene knockout screen with 11 bp deletion in wnt16, which led to the premature termination of amino acid translation and significantly reduced wnt16 expression, thus obtaining the wnt16-/- zebrafish model. The expression of wnt16 in bone-related parts was detected via in situ hybridization. The head, spine, and tail exhibited significant deformities, and the bone mineral density and trabecular bone decreased in wnt16-/- using light microscopy and micro-CT analysis. RNA sequencing was performed to explore the differentially expressed genes (DEGs). Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis found that the down-regulated DEGs are mainly concentrated in mTOR, FoxO, and VEGF pathways. Protein–protein interaction (PPI) network analysis was performed with the detected DEGs. Eight down-regulated DEGs including akt1, bnip4, ptena, vegfaa, twsg1b, prkab1a, prkab1b, and pla2g4f.2 were validated by qRT-PCR and the results were consistent with the RNA-seq data. Overall, our work provides key insights into the influence of wnt16 gene on skeletal development.

Micro‐CT analysis of the Lisfranc complex reveals higher bone mineral density in dorsal compared to plantar regions

2021 ◽  
Author(s):  
Melissa R. Requist ◽  
Yantarat Sripanich ◽  
Tim Rolvien ◽  
Amy L. Lenz ◽  
Alexej Barg
Keyword(s):  
Bone Mineral Density ◽  
Bone Mineral ◽  
Micro Ct ◽  
Mineral Density ◽  
Ct Analysis

scholarly journals Role of the lncRNA BACE1-AS in shared disease mechanisms between heart failure and Alzheimer's disease

2020 ◽  
Vol 41 (Supplement_2) ◽  
Author(s):  
S Greco ◽  
A Made' ◽  
A.S Tascini ◽  
J Garcia Manteiga ◽  
S Castelvecchio ◽  
...  
Keyword(s):  
Heart Failure ◽  
Alzheimer’S Disease ◽  
Alzheimer's Disease ◽  
In Situ Hybridization ◽  
Cell Apoptosis ◽  
Common Disease ◽  
Funding Source ◽  
Mrna Levels ◽  
Rna Seq

Abstract Background BACE1 encodes for β-secretase, the key enzyme involved in β-amyloid (βA) generation, a peptide well known for its involvement in Alzheimer's disease (AD). Of note, heart failure (HF) and AD share several risk factors and effectors. We recently showed that, in the heart of ischemic HF patients, the levels of both BACE1, its antisense RNA BACE1-AS and βA are all increased. BACE1-AS positively regulates the expression of BACE1, triggering βA intracellular accumulation, and its overexpression or βA administration induce cardiovascular-cell apoptosis. Aim To characterize the transcripts of the BACE1 locus and to investigate the molecular mechanisms underpinning BACE1-AS regulation of cell vitality. Methods By PCR and sequencing, we studied in the heart the expression of a variety of antisense BACE1 transcripts predicted by FANTOM CAT Epigenome. We studied BACE1 RNA stability by BrdU pulse chase experiments (BRIC assay). The cellular localization of BACE1-AS RNA was investigated by in situ hybridization assay. BACE1-AS binding RNAs were evaluated by BACE1-AS-MS2-Tag pull-down in AC16 cardiomyocytes followed by RNA-seq. Enriched RNAs were validated by qPCR and analysed by bioinformatics comparison with publicly available gene expression datasets of AD brains. Results We readily detected several antisense BACE1 transcripts expressed in AC16 cardiomyocytes; however, only BACE1-AS RNAs overlapping exon 6 of BACE1 positively regulated BACE1 mRNA levels, acting by increasing its stability. BACE1 silencing reverted cell apoptosis induced by BACE1-AS expression, indicating that BACE1 is a functional target of BACE1-AS. However, in situ hybridization experiments indicated a mainly nuclear localization for BACE1-AS, which displayed a punctuated distribution, compatible with chromatin association and indicative of potential additional targets. To identify other BACE1-AS binding RNAs, a BACE1-AS-MS2-tag pull-down was performed and RNA-seq of the enriched RNAs identified 698 BACE1-AS interacting RNAs in cardiomyocytes. Gene ontology of the BACE1-AS binding RNAs identified categories of relevance for cardiovascular or neurological diseases, such as dopaminergic synapse, glutamatergic synapse, calcium signalling pathway and voltage-gated channel activity. In spite of the differences between brain and heart transcriptomes, BACE1-AS-interacting RNAs identified in cardiomyocytes were significantly enriched in transcripts differentially expressed in AD brains as well as in RNAs expressed by enhancer genomic regions that are significantly hypomethylated in AD brains. Conclusions These data shed a new light on the complexity of BACE1-AS locus and on the existence of RNAs interacting with BACE1-AS with a potential as enhancer-RNAs. Moreover, the dysregulation of the BACE1-AS/BACE1/βA pathway may be a common disease mechanism shared by cardiovascular and neurological degenerative diseases. Funding Acknowledgement Type of funding source: Public grant(s) – National budget only. Main funding source(s): Italian Health Ministery_Ricerca Corrente 2020

scholarly journals In situ RNA-seq

2013 ◽  
Vol 14 (9) ◽  
pp. 596-596 ◽  
Author(s):  
Hannah Stower
Keyword(s):  
Rna Seq

scholarly journals AnIn VivoZebrafish Model for Interrogating ROS-Mediated Pancreaticβ-Cell Injury, Response, and Prevention

2018 ◽  
Vol 2018 ◽  
pp. 1-8 ◽  
Author(s):  
Abhishek A. Kulkarni ◽  
Abass M. Conteh ◽  
Cody A. Sorrell ◽  
Anjali Mirmira ◽  
Sarah A. Tersey ◽  
...  
Keyword(s):  
Cell Death ◽  
Caspase 3 ◽  
Cell Injury ◽  
Zebrafish Model ◽  
Chemical Genetic ◽  
Regulated Cell Death ◽  
Specific Manner ◽  
High Doses

It is well known that a chronic state of elevated reactive oxygen species (ROS) in pancreaticβ-cells impairs their ability to release insulin in response to elevated plasma glucose. Moreover, at its extreme, unmitigated ROS drives regulated cell death. This dysfunctional state of ROS buildup can result both from genetic predisposition and environmental factors such as obesity and overnutrition. Importantly, excessive ROS buildup may underlie metabolic pathologies such as type 2 diabetes mellitus. The ability to monitor ROS dynamics inβ-cells in situ and to manipulate it via genetic, pharmacological, and environmental means would accelerate the development of novel therapeutics that could abate this pathology. Currently, there is a lack of models with these attributes that are available to the field. In this study, we use a zebrafish model to demonstrate that ROS can be generated in aβ-cell-specific manner using a hybrid chemical genetic approach. Using a transgenic nitroreductase-expressing zebrafish line,Tg(ins:Flag-NTR)s950, treated with the prodrug metronidazole (MTZ), we found that ROS is rapidly and explicitly generated inβ-cells. Furthermore, the level of ROS generated was proportional to the dosage of prodrug added to the system. At high doses of MTZ, caspase 3 was rapidly cleaved,β-cells underwent regulated cell death, and macrophages were recruited to the islet to phagocytose the debris. Based on our findings, we propose a model for the mechanism of NTR/MTZ action in transgenic eukaryotic cells and demonstrate the robust utility of this system to model ROS-related disease pathology.

scholarly journals Local Changes to the Distal Femoral Growth Plate Following Injury in Mice

2017 ◽  
Vol 139 (7) ◽  
Author(s):  
Lauren M. Mangano Drenkard ◽  
Meghan E. Kupratis ◽  
Katie Li ◽  
Louis C. Gerstenfeld ◽  
Elise F. Morgan
Keyword(s):  
Growth Plate ◽  
Skeletal Development ◽  
Global Changes ◽  
Mineral Density ◽  
Injury Site ◽  
Hypertrophic Zone ◽  
Proliferative Zone ◽  
Spatial Changes ◽  
Zonal Organization ◽  
Growth Plate Injury

Injury to the growth plate is associated with growth disturbances, most notably premature cessation of growth. The goal of this study was to identify spatial changes in the structure and composition of the growth plate in response to injury to provide a foundation for developing therapies that minimize the consequences for skeletal development. We used contrast-enhanced microcomputed tomography (CECT) and histological analyses of a murine model of growth plate injury to quantify changes in the cartilaginous and osseous tissue of the growth plate. To distinguish between local and global changes, the growth plate was divided into regions of interest near to and far from the injury site. We noted increased thickness and CECT attenuation (a measure correlated with glycosaminoglycan (GAG) content) near the injury, and increased tissue mineral density (TMD) of bone bridges within the injury site, compared to outside the injury site and contralateral growth plates. Furthermore, we noted disruption of the normal zonal organization of the physis. The height of the hypertrophic zone was increased at the injury site, and the relative height of the proliferative zone was decreased across the entire injured growth plate. These results indicate that growth plate injury leads to localized disruption of cellular activity and of endochondral ossification. These local changes in tissue structure and composition may contribute to the observed retardation in femur growth. In particular, the changes in proliferative and hypertrophic zone heights seen following injury may impact growth and could be targeted when developing therapies for growth plate injury.

scholarly journals The ciliary protein IFT88 controls post-natal cartilage thickness and influences development of osteoarthritis

2020 ◽  
Author(s):  
CR Coveney ◽  
L Zhu ◽  
J Miotla-Zarebska ◽  
B Stott ◽  
I Parisi ◽  
...  
Keyword(s):  
Articular Cartilage ◽  
Cell Biology ◽  
Articular Chondrocytes ◽  
Skeletal Development ◽  
Cartilage Degradation ◽  
Cartilage Thickness ◽  
Tissue Growth ◽  
Calcified Cartilage ◽  
Health And Disease

AbstractMechanical forces are known to drive cellular signalling programmes in cartilage development, health, and disease. Proteins of the primary cilium, implicated in mechanoregulation, control cartilage formation during skeletal development, but their role in post-natal cartilage is unknown. Ift88fl/fl and AggrecanCreERT2 mice were crossed to create a cartilage specific inducible knockout mouse AggrecanCreERT2;Ift88fl/fl. Tibial articular cartilage thickness was assessed, through adolescence and adulthood, by histomorphometry and integrity by OARSI score. In situ cell biology was investigated by immunohistochemistry (IHC) and qPCR of micro-dissected cartilage. OA was induced by destabilisation of the medial meniscus (DMM). Some mice were provided with exercise wheels in their cage. Deletion of IFT88 resulted in a reduction in medial articular cartilage thickness (atrophy) during adolescence from 102.57μm, 95% CI [94.30, 119.80] in control (Ift88fl/fl) to 87.36μm 95% CI [81.35, 90.97] in AggrecanCreERT2;Ift88fl/fl by 8-weeks p<0.01, and adulthood (104.00μm, 95% CI [100.30, 110.50] in Ift88fl/fl to 89.42μm 95% CI [84.00, 93.49] in AggrecanCreERT2;Ift88fl/fl, 34-weeks, p<0.0001) through a reduction in calcified cartilage. Thinning in adulthood was associated with spontaneous cartilage degradation. Following DMM, AggrecanCreERT2;Ift88fl/fl mice had increased OA (OARSI scores at 12 weeks Ift88fl/fl = 22.08 +/− 9.30, and AggrecanCreERT2;Ift88fl/fl = 29.83 +/− 7.69). Atrophy was not associated with aggrecanase-mediated destruction or chondrocyte hypertrophy. Ift88 expression positively correlated with Tcf7l2 and connective tissue growth factor. Cartilage thickness was restored in AggrecanCreERT2;Ift88fl/fl by voluntary wheel exercise. Our results demonstrate that ciliary IFT88 regulates cartilage thickness and is chondroprotective, potentially through modulating mechanotransduction pathways in articular chondrocytes.

scholarly journals circHMGCS1–016 reshapes immune environment by sponging miR-1236-3p to regulate CD73 and GAL-8 expression in intrahepatic cholangiocarcinoma

2021 ◽  
Vol 40 (1) ◽  
Author(s):  
Ya-Ping Xu ◽  
Ze-Ning Dong ◽  
Si-Wei Wang ◽  
Yi-Min Zheng ◽  
Chi Zhang ◽  
...  
Keyword(s):  
In Situ Hybridization ◽  
Intrahepatic Cholangiocarcinoma ◽  
Molecular Mechanisms ◽  
Prognostic Significance ◽  
Luciferase Reporter ◽  
Mouse Tumor ◽  
Rna Seq ◽  
Potential Biomarker

Abstract Background Accumulating evidence indicates that circRNAs may serve as essential regulators in the progression of several human cancers, but the function and mechanism of circRNAs in intrahepatic cholangiocarcinoma (ICC) are largely unknown. Methods RNA-seq was used to assess differentially expressed circRNAs between 4 ICC and peritumor tissues. Quantitative RT-PCR and in situ hybridization were used to determine the circHMGCS1–016 expression in ICC tissues. The function and mechanism of circHMGCS1–016 were further identified via in vivo experiments. The clinical characteristics and prognostic significance of circHMGCS1–016 were analyzed by a retrospective study. The functions of circHMGCS1–016 were assessed via modifying circRNA expression in ICC cells. Moreover, the molecular mechanisms of circHMGCS1–016 in ICC cells were explored by circRNA precipitation, miRNA immunoprecipitation, SILAC and luciferase reporter assays. Results We identified that compared with peritumor tissues, ICC tissues expressed hsa_circ_0008621 (circHMGCS1–016) high by RNA-seq, which was further identified by qRT-PCR and in situ hybridization. Moreover, the expression of circHMGCS1–016 was revealed to be associated with survival and recurrence of ICC patients. By regulating circHMGCS1–016 expression, we found that elevated circHMGCS1–016 promoted ICC development both in vitro and in vivo. By SILAC and circRNA-pull down, we demonstrated that circHMGCS1–016 induced ICC cell invasion and reshaped the tumor immune microenvironment via the miR-1236-3p/CD73 and GAL-8 axis. In ICC tissues, we uncovered that a high level of circHMGCS1–016 was positively associated with CD73 and GAL-8 expression and negatively related to the CD8+ T cells infiltration, which was further validated by establishing a humanized mouse tumor model. Importantly, we displayed that ICC patients with high levels of circHMGCS1–016 in tumor tissues benefited less from anti-PD1 treatment compared to those with low levels of circHMGCS1–016. Conclusions CircHMGCS1–016 is a forceful contributor in ICC development and immune tolerance via miR-1236-3p/CD73 and GAL-8 axis. CircHMGCS1–016 can be explored as a new potential biomarker and therapeutic target for PD1-resistant ICC.

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