Abstract 283: Nppa Marks A Subset Of Embryonic Cardiomyocytes Predestined To Form Trabeculae In Zebrafish

2014 ◽  
Vol 115 (suppl_1) ◽  
Author(s):  
Rima Arnaout ◽  
Didier Y Stainier ◽  
Shaun R Coughlin
Keyword(s):  
Zebrafish Embryo ◽  
Transgenic Zebrafish ◽  
Loss Of Function ◽  
Atrial Contraction ◽  
Embryonic Cardiomyocytes ◽  
External Development ◽  
Insight Into ◽  
Spinning Disc ◽  
Atrial Myosin

The development and maintenance of cardiac trabeculae is required for proper ventricular function. Aberrant trabeculation can cause heart failure, arrhythmia, and death. Yet the mechanisms controlling initiation of trabeculation are incompletely understood, partly due to a lack of markers for trabeculation during early embryogenesis. We hypothesized that natriuretic peptide A (nppa) marks trabeculae before cardiomyocytes leave the single-celled ventricular layer. While nppa has been studied in mammals, the external development of the translucent zebrafish embryo offers new insight into the earliest stages of trabeculation. We have found by in situ hybridization, and by creating a fluorescent transgenic zebrafish line, that nppa exclusively marks zebrafish cardiac trabeculae from their onset through adulthood. Live spinning disc confocal video microscopy shows that nppa:GFP-positive cells move into the trabecular layer starting at 60 hours post fertilization (hpf). GFP-positive trabeculae are seen by 5 days post fertilization (dpf) in normal larvae. However, in cardiac troponin T2a mutants and in erbB2 mutants _ both known not to trabeculate _ nppa is still expressed, but the nppa:GFP positive cells fail to form trabecular projections. In embryos injected with a morpholino against atrial myosin heavy chain 6, where weakened atrial contraction leads to weaker blood flow without affecting the ventricle directly, GFP-positive cells form trabeculae similar to uninjected controls. Using Clustered Regularly Interspaced Short Palindromic Repeats (CRISPRs), we created nppa mutants causing frameshift deletions. Mutants have poorly contractile hearts at 3 dpf and have been crossed into transgenic reporter lines in order to specifically assess trabeculation. Together, these data suggest that nppa marks a subset of embryonic cardiomyocytes destined to form trabeculae, and that nppa loss of function causes cardiac defects.

scholarly journals The CD33 short isoform is a gain-of-function variant that enhances Aβ1–42 phagocytosis in microglia

2021 ◽  
Vol 16 (1) ◽  
Author(s):  
Abhishek Bhattacherjee ◽  
Jaesoo Jung ◽  
Sameera Zia ◽  
Madelene Ho ◽  
Ghazaleh Eskandari-Sedighi ◽  
...  
Keyword(s):  
Cell Lineage ◽  
Strong Support ◽  
U937 Cells ◽  
Loss Of Function ◽  
Primary Microglia ◽  
Gain Of Function ◽  
Intracellular Location ◽  
Insight Into

Abstract Background CD33 is genetically linked to Alzheimer’s disease (AD) susceptibility through differential expression of isoforms in microglia. The role of the human CD33 short isoform (hCD33m), preferentially encoded by an AD-protective CD33 allele (rs12459419T), is unknown. Here, we test whether hCD33m represents a loss-of-function or gain-of-function variant. Methods We have developed two models to test the role of hCD33m. The first is a new strain of transgenic mice expressing hCD33m in the microglial cell lineage. The second is U937 cells where the CD33 gene was disrupted by CRISPR/Cas9 and complemented with different variants of hCD33. Primary microglia and U937 cells were tested in phagocytosis assays and single cell RNA sequencing (scRNAseq) was carried out on the primary microglia. Furthermore, a new monoclonal antibody was developed to detect hCD33m more efficiently. Results In both primary microglia and U937 cells, we find that hCD33m enhances phagocytosis. This contrasts with the human CD33 long isoform (hCD33M) that represses phagocytosis, as previously demonstrated. As revealed by scRNAseq, hCD33m+ microglia are enriched in a cluster of cells defined by an upregulated expression and gene regulatory network of immediate early genes, which was further validated within microglia in situ. Using a new hCD33m-specific antibody enabled hCD33m expression to be examined, demonstrating a preference for an intracellular location. Moreover, this newly discovered gain-of-function role for hCD33m is dependent on its cytoplasmic signaling motifs, dominant over hCD33M, and not due to loss of glycan ligand binding. Conclusions These results provide strong support that hCD33m represents a gain-of-function isoform and offers insight into what it may take to therapeutically capture the AD-protective CD33 allele.

scholarly journals Knockdown of Laminin gamma-3 (Lamc3) impairs motoneuron guidance in the zebrafish embryo

2017 ◽  
Vol 2 ◽  
pp. 111
Author(s):  
Alexander M. J. Eve ◽  
James C. Smith
Keyword(s):  
Endothelial Cells ◽  
Zebrafish Embryo ◽  
Axonal Guidance ◽  
Loss Of Function ◽  
Cardinal Vein ◽  
Morpholino Oligonucleotides ◽  
Antisense Morpholino Oligonucleotides ◽  
Antisense Morpholino

Background: Previous work in the zebrafish embryo has shown that laminin γ-3 (lamc3) is enriched in endothelial cells marked by expression of fli1a, but the role of Lamc3 has been unknown. Methods: We use antisense morpholino oligonucleotides, and CRISPR/Cas9 mutagenesis of F0 embryos, to create zebrafish embryos in which lamc3 expression is compromised. Transgenic imaging, immunofluorescence, and in situ hybridisation reveal that Lamc3 loss-of-function affects the development of muscle pioneers, endothelial cells, and motoneurons. Results: Lamc3 is enriched in endothelial cells during zebrafish development, but it is also expressed by other tissues. Depletion of Lamc3 by use of antisense morpholino oligonucleotides perturbs formation of the parachordal chain and subsequently the thoracic duct, but Lamc3 is not required for sprouting of the cardinal vein. F0 embryos in which lamc3 expression is perturbed by a CRISPR/Cas9 approach also fail to form a parachordal chain, but we were unable to establish a stable lamc3 null line. Lamc3 is dispensable for muscle pioneer specification and for the expression of netrin-1a in these cells. Lamc3 knockdown causes netrin-1a up-regulation in the neural tube and there is increased Netrin-1 protein throughout the trunk of the embryo. Axonal guidance of rostral primary motoneurons is defective in Lamc3 knockdown embryos. Conclusions: We suggest that knockdown of Lamc3 perturbs migration of rostral primary motoneurons at the level of the horizontal myoseptum, indicating that laminin γ3 plays a role in motoneuron guidance.

scholarly journals Pitx controls amphioxus asymmetric morphogenesis by promoting left-side development and repressing right-side formation

BMC Biology ◽  
2021 ◽  
Vol 19 (1) ◽  
Author(s):  
Chaofan Xing ◽  
Rongrong Pan ◽  
Guangwei Hu ◽  
Xian Liu ◽  
Yiquan Wang ◽  
...  
Keyword(s):  
Binding Sites ◽  
Essential Feature ◽  
Loss Of Function ◽  
Partial Loss ◽  
Developmental Mechanism ◽  
Right Isomerism ◽  
The Right ◽  
Insight Into ◽  
Made In

Abstract Background Left-right (LR) asymmetry is an essential feature of bilateral animals. Studies in vertebrates show that LR asymmetry formation comprises three major steps: symmetry breaking, asymmetric gene expression, and LR morphogenesis. Although much progress has been made in the first two events, mechanisms underlying asymmetric morphogenesis remain largely unknown due to the complex developmental processes deployed by vertebrate organs. Results We here addressed this question by studying Pitx gene function in the basal chordate amphioxus whose asymmetric organogenesis, unlike that in vertebrates, occurs essentially in situ and does not rely on cell migration. Pitx null mutation in amphioxus causes loss of all left-sided organs and incomplete ectopic formation of all right-sided organs on the left side, whereas Pitx partial loss-of-function leads to milder phenotypes with only some LR organs lost or ectopically formed. At the N1 to N3 stages, Pitx expression is gradually expanded from the dorsal anterior domain to surrounding regions. This leads to activation of genes like Lhx3 and/or Prop1 and Pit, which are essential for left-side organs, and downregulation of genes like Hex and/or Nkx2.1 and FoxE4, which are required for right-side organs to form ectopically on the left side. In Pitx mutants, the left-side expressed genes are not activated, while the right-side genes fail to decrease expression on the left side. In contrast, in embryos overexpressing Pitx genes, the left-side genes are induced ectopically on the right side, and the right-side genes are inhibited. Several Pitx binding sites are identified in the upstream sequences of the left-side and right-side genes which are essential for activation of the former and repression of the latter by Pitx. Conclusions Our results demonstrate that (1) Pitx is a major (although not the only) determinant of asymmetric morphogenesis in amphioxus, (2) the development of different LR organs have distinct requirements for Pitx activity, and (3) Pitx controls amphioxus LR morphogenesis probably through inducing left-side organs and inhibiting right-side organs directly. These findings show much more dependence of LR organogenesis on Pitx in amphioxus than in vertebrates. They also provide insight into the molecular developmental mechanism of some vertebrate LR organs like the lungs and atria, since they show a right-isomerism phenotype in Pitx2 knockout mice like right-sided organs in Pitx mutant amphioxus. Our results also explain why some organs like the adenohypophysis are asymmetrically located in amphioxus but symmetrically positioned in vertebrates.

scholarly journals Development of Fragility Curves for Piping and Slope Stability of River Levees

Water ◽  
2021 ◽  
Vol 13 (5) ◽  
pp. 738
Author(s):  
Nicola Rossi ◽  
Mario Bačić ◽  
Meho Saša Kovačević ◽  
Lovorka Librić
Keyword(s):  
Slope Stability ◽  
Fragility Curves ◽  
Safety Margin ◽  
Water Levels ◽  
Flood Event ◽  
Soil Parameters ◽  
Design Code ◽  
A Site ◽  
Insight Into

The design code Eurocode 7 relies on semi-probabilistic calculation procedures, through utilization of the soil parameters obtained by in situ and laboratory tests, or by the means of transformation models. To reach a prescribed safety margin, the inherent soil parameter variability is accounted for through the application of partial factors to either soil parameters directly or to the resistance. However, considering several sources of geotechnical uncertainty, including the inherent soil variability, measurement error and transformation uncertainty, full probabilistic analyses should be implemented to directly consider the site-specific variability. This paper presents the procedure of developing fragility curves for levee slope stability and piping as failure mechanisms that lead to larger breaches, where a direct influence of the flood event intensity on the probability of failure is calculated. A range of fragility curve sets is presented, considering the variability of levee material properties and varying durations of the flood event, thus providing crucial insight into the vulnerability of the levee exposed to rising water levels. The procedure is applied to the River Drava levee, a site which has shown a continuous trend of increased water levels in recent years.

Insight into the enhanced photocatalytic properties of AgBr/Ag4P2O7 composites synthesized via in situ ion exchange reaction

2021 ◽  
Vol 9 (1) ◽  
pp. 104889
Author(s):  
Wyllamanney da S. Pereira ◽  
Fabrício B. Destro ◽  
Cipriano B. Gozzo ◽  
Edson R. Leite ◽  
Júlio C. Sczancoski
Keyword(s):  
Ion Exchange ◽  
Exchange Reaction ◽  
Photocatalytic Properties ◽  
Ion Exchange Reaction ◽  
Insight Into

scholarly journals In situ synthesis of methane using Ag–GDC composite electrodes in a tubular solid oxide electrolytic cell: new insight into the role of oxide ion removal

2021 ◽  
Vol 5 (7) ◽  
pp. 2055-2064
Author(s):  
Saheli Biswas ◽  
Aniruddha P. Kulkarni ◽  
Daniel Fini ◽  
Sarbjit Giddey ◽  
Sankar Bhattacharya
Keyword(s):  
Electrolytic Cell ◽  
Composite Electrodes ◽  
Ion Removal ◽  
Single Temperature ◽  
Methanation Catalyst ◽  
Oxide Ion ◽  
Insight Into ◽  
Electrochemical Phenomenon

In situ synthesis of methane in a single-temperature zone SOEC in the absence of any methanation catalyst is a completely electrochemical phenomenon governed by the thermodynamic equilibrium of various reactions.

In situ loading of well-dispersed silver nanoparticles on nanocrystalline magnesium oxide for real-time monitoring of catalytic reactions by surface enhanced Raman spectroscopy

Nanoscale ◽  
2015 ◽  
Vol 7 (40) ◽  
pp. 16952-16959 ◽  
Author(s):  
Kaige Zhang ◽  
Gongke Li ◽  
Yuling Hu
Keyword(s):  
Raman Spectroscopy ◽  
Surface Enhanced Raman Spectroscopy ◽  
Catalytic Reactions ◽  
Reaction Intermediates ◽  
Reaction Conditions ◽  
Surface Enhanced ◽  
Surface Enhanced Raman ◽  
Nanocrystalline Magnesium Oxide ◽  
Insight Into

The surface-enhanced Raman spectroscopy (SERS) technique is of great importance for insight into the transient reaction intermediates and mechanistic pathways involved in heterogeneously catalyzed chemical reactions under actual reaction conditions, especially in water.

In situ electrochemical microcantilever bending test: A new insight into hydrogen enhanced cracking

2017 ◽  
Vol 132 ◽  
pp. 17-21 ◽  
Author(s):  
Tarlan Hajilou ◽  
Yun Deng ◽  
Bjørn Rune Rogne ◽  
Nousha Kheradmand ◽  
Afrooz Barnoush
Keyword(s):  
Bending Test ◽  
Insight Into
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