Tek (Tie2) is not required for cardiovascular development in zebrafish

ABSTRACTAngiopoietin/TIE signalling plays a major role in blood and lymphatic vessel development. In mouse, Tek (previously known as Tie2) mutants die prenatally due to a severely underdeveloped cardiovascular system. In contrast, in zebrafish, previous studies have reported that although embryos injected with tek morpholinos (MOs) exhibit severe vascular defects, tek mutants display no obvious vascular malformations. To further investigate the function of zebrafish Tek, we generated a panel of loss-of-function tek mutants, including RNA-less alleles, an allele lacking the MO-binding site, an in-frame deletion allele and a premature termination codon-containing allele. Our data show that all these mutants survive to adulthood with no obvious cardiovascular defects. MO injections into tek mutants lacking the MO-binding site or the entire tek locus cause similar vascular defects to those observed in MO-injected +/+ siblings, indicating off-target effects of the MOs. Surprisingly, comprehensive phylogenetic profiling and synteny analyses reveal that Tek was lost in the largest teleost clade, suggesting a lineage-specific shift in the function of TEK during vertebrate evolution. Altogether, these data show that Tek is dispensable for zebrafish development, and probably dispensable in most teleost species.

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Knockdown of Novel lncRNA TCONS_00028652 in Zebrafish Affects Embryonic Vasculature Development

International Journal of Agriculture and Biology ◽

10.17957/ijab/15.1735 ◽

2021 ◽

Vol 25 (04) ◽

pp. 831-837

Author(s):

Chune Zhou

Keyword(s):

Embryonic Development ◽

Molecular Mechanisms ◽

Marker Gene ◽

Transgenic Fish ◽

Cardiovascular Development ◽

Loss Of Function ◽

Flanking Sequence ◽

Real Time Quantitative Pcr ◽

Vessel Development ◽

Coding Potential

Long non-coding RNAs (lncRNA) are increasingly being regard as potential key regulators of biological process, however, little is known about the function of most of them. The involvement of a novel lncRNA (ENSDART000000150571), previously named TCONS_00028652, in intersegmental vessel development was investigated in this study. TCONS_00028652, having a single exon (568 bp), is located in chromosome 16 at position from 15786804 to 15786237, which was previously identified as an embryonic and adult heart-enrichedlncRNA. Bioinformatics analysis and annotation of TCONS_00028652 was performed using online databases. Its protein-coding potential was assessed using online softwares: the Coding Potential Assessing Tool (CPAT) and the Coding Potential Calculator (CPC). To verify its real existence, a cloned fragment was proliferated by designing primers against 5’ and 3’ exon-flanking sequence. Subsequently, spatiotemporal expression during zebrafish embryonic development was determined using real-time quantitative PCR (qPCR) and whole mount in situ hybridization. We found that lncRNA TCONS_00028652 was generally expressed throughout early stages of zebrafish embryonic development and predominantly in embryonic brain, tail, and heart. Knockdown of TCONS_00028652 using morpholino oligonucleotides (MO) resulted in intersegmental vessel defects, suggesting that TCONS_00028652 is indispensable for embryonic vascular development in zebrafish. Using Tg (flil:EGFP) transgenic fish expressing a cardiovascular marker gene, loss of function experiments confirmed that TCONS_00028652 was involved in embryonic intersegmental vessel development. Our results may lead to valuable understanding of lncRNAs functions in zebrafish embryonic development and molecular mechanisms of embryonic cardiovascular development. © 2021 Friends Science Publishers

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Faculty Opinions recommendation of lyve1 expression reveals novel lymphatic vessels and new mechanisms for lymphatic vessel development in zebrafish.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.717597809.793002865 ◽

2012 ◽

Author(s):

Peter Koopman ◽

Mathias Francois

Keyword(s):

Lymphatic Vessel ◽

Lymphatic Vessels ◽

Vessel Development

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Faculty Opinions recommendation of Isl1-expressing non-venous cell lineage contributes to cardiac lymphatic vessel development.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.735802869.793565952 ◽

2019 ◽

Author(s):

Guillermo Oliver ◽

Xiaolei Liu

Keyword(s):

Lymphatic Vessel ◽

Cell Lineage ◽

Vessel Development

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Maternal iron deficiency perturbs embryonic cardiovascular development in mice

Nature Communications ◽

10.1038/s41467-021-23660-5 ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Jacinta I. Kalisch-Smith ◽

Nikita Ved ◽

Dorota Szumska ◽

Jacob Munro ◽

Michael Troup ◽

...

Keyword(s):

Iron Deficiency ◽

Environment Interaction ◽

Cardiovascular Development ◽

Retinoic Acid Signalling ◽

Gene Environment Interaction ◽

Iron Administration ◽

Gene Environment ◽

Cardiovascular Defects ◽

Craniofacial Defects ◽

Maternal Iron

AbstractCongenital heart disease (CHD) is the most common class of human birth defects, with a prevalence of 0.9% of births. However, two-thirds of cases have an unknown cause, and many of these are thought to be caused by in utero exposure to environmental teratogens. Here we identify a potential teratogen causing CHD in mice: maternal iron deficiency (ID). We show that maternal ID in mice causes severe cardiovascular defects in the offspring. These defects likely arise from increased retinoic acid signalling in ID embryos. The defects can be prevented by iron administration in early pregnancy. It has also been proposed that teratogen exposure may potentiate the effects of genetic predisposition to CHD through gene–environment interaction. Here we show that maternal ID increases the severity of heart and craniofacial defects in a mouse model of Down syndrome. It will be important to understand if the effects of maternal ID seen here in mice may have clinical implications for women.

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Cross-Species RNAi Rescue Platform in Drosophila melanogaster

Genetics ◽

10.1534/genetics.109.106567 ◽

2009 ◽

Vol 183 (3) ◽

pp. 1165-1173 ◽

Cited By ~ 34

Author(s):

Shu Kondo ◽

Matthew Booker ◽

Norbert Perrimon

Keyword(s):

Cell Culture ◽

Drosophila Melanogaster ◽

Large Scale ◽

Transgenic Animals ◽

Selection Marker ◽

Bacterial Artificial Chromosomes ◽

Loss Of Function ◽

Artificial Chromosomes ◽

Target Effects

RNAi-mediated gene knockdown in Drosophila melanogaster is a powerful method to analyze loss-of-function phenotypes both in cell culture and in vivo. However, it has also become clear that false positives caused by off-target effects are prevalent, requiring careful validation of RNAi-induced phenotypes. The most rigorous proof that an RNAi-induced phenotype is due to loss of its intended target is to rescue the phenotype by a transgene impervious to RNAi. For large-scale validations in the mouse and Caenorhabditis elegans, this has been accomplished by using bacterial artificial chromosomes (BACs) of related species. However, in Drosophila, this approach is not feasible because transformation of large BACs is inefficient. We have therefore developed a general RNAi rescue approach for Drosophila that employs Cre/loxP-mediated recombination to rapidly retrofit existing fosmid clones into rescue constructs. Retrofitted fosmid clones carry a selection marker and a phiC31 attB site, which facilitates the production of transgenic animals. Here, we describe our approach and demonstrate proof-of-principle experiments showing that D. pseudoobscura fosmids can successfully rescue RNAi-induced phenotypes in D. melanogaster, both in cell culture and in vivo. Altogether, the tools and method that we have developed provide a gold standard for validation of Drosophila RNAi experiments.

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Specificity of RNAi, LNA and CRISPRi as loss-of-function methods in transcriptional analysis

10.1101/234930 ◽

2017 ◽

Cited By ~ 1

Author(s):

Lovorka Stojic ◽

Aaron Lun ◽

Jasmin Mangei ◽

Patrice Mascalchi ◽

Valentina Quarantotti ◽

...

Keyword(s):

Antisense Oligonucleotides ◽

Noncoding Rna ◽

Specific Activity ◽

Transcriptional Analysis ◽

Clonal Variation ◽

Loss Of Function ◽

Negative Controls ◽

Whole Transcriptome ◽

Target Effects

ABSTRACTLoss-of-function (LOF) methods, such as RNA interference (RNAi), antisense oligonucleotides or CRISPR-based genome editing, provide unparalleled power for studying the biological function of genes of interest. When coupled with transcriptomic analyses, LOF methods allow researchers to dissect networks of transcriptional regulation. However, a major concern is nonspecific targeting, which involves depletion of transcripts other than those intended. The off-target effects of each of these common LOF methods have yet to be compared at the whole-transcriptome level. Here, we systematically and experimentally compared non-specific activity of RNAi, antisense oligonucleotides and CRISPR interference (CRISPRi). All three methods yielded non-negligible offtarget effects in gene expression, with CRISPRi exhibiting clonal variation in the transcriptional profile. As an illustrative example, we evaluated the performance of each method for deciphering the role of a long noncoding RNA (lncRNA) with unknown function. Although all LOF methods reduced expression of the candidate lncRNA, each method yielded different sets of differentially expressed genes upon knockdown as well as a different cellular phenotype. Therefore, to definitively confirm the functional role of a transcriptional regulator, we recommend the simultaneous use of at least two different LOF methods and the inclusion of multiple, specifically designed negative controls.

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Improved analysis of CRISPR fitness screens and reduced off-target effects with the BAGEL2 gene essentiality classifier

10.1101/2020.05.30.125526 ◽

2020 ◽

Cited By ~ 2

Author(s):

Eiru Kim ◽

Traver Hart

Keyword(s):

Dynamic Range ◽

Quality Data ◽

Loss Of Function ◽

Genetic Screens ◽

Gene Essentiality ◽

Guide Rna ◽

Genome Wide ◽

Improved Model ◽

Target Effects

AbstractIdentifying essential genes in genome-wide loss of function screens is a critical step in functional genomics and cancer target finding. We previously described the Bayesian Analysis of Gene Essentiality (BAGEL) algorithm for accurate classification of gene essentiality from short hairpin RNA and CRISPR/Cas9 genome wide genetic screens. Here, we introduce an updated version, BAGEL2, which employs an improved model that offers greater dynamic range of Bayes Factors, enabling detection of tumor suppressor genes, and a multi-target correction that reduces false positives from off-target CRISPR guide RNA. We also suggest a metric for screen quality at the replicate level and demonstrate how different algorithms handle lower-quality data in substantially different ways. BAGEL2 is written in Python 3 and source code, along with all supporting files, are available on github (https://github.com/hart-lab/bagel).

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Fgf8is required for pharyngeal arch and cardiovascular development in the mouse

Development ◽

10.1242/dev.129.19.4613 ◽

2002 ◽

Vol 129 (19) ◽

pp. 4613-4625 ◽

Cited By ~ 15

Author(s):

Radwan Abu-Issa ◽

Graham Smyth ◽

Ida Smoak ◽

Ken-ichi Yamamura ◽

Erik N. Meyers

Keyword(s):

Craniofacial Development ◽

Mouse Embryos ◽

Pharyngeal Arch ◽

Compound Heterozygous ◽

Cardiovascular Development ◽

Early Analysis ◽

Pharyngeal Arches ◽

The Face ◽

Cardiovascular Defects ◽

Additional Role

We present here an analysis of cardiovascular and pharyngeal arch development in mouse embryos hypomorphic for Fgf8. Previously, we have described the generation of Fgf8 compound heterozygous (Fgf8neo/–) embryos. Although early analysis demonstrated that some of these embryos have abnormal left-right (LR) axis specification and cardiac looping reversals, the number and type of cardiac defects present at term suggested an additional role for Fgf8 in cardiovascular development. Most Fgf8neo/– mutant embryos survive to term with abnormal cardiovascular patterning, including outflow tract, arch artery and intracardiac defects. In addition, these mutants have hypoplastic pharyngeal arches, small or absent thymus and abnormal craniofacial development. Neural crest cells (NCCs) populate the pharyngeal arches and contribute to many structures of the face, neck and cardiovascular system, suggesting that Fgf8 may be required for NCC development. Fgf8 is expressed within the developing pharyngeal arch ectoderm and endoderm during NCC migration through the arches. Analysis of NCC development in Fgf8neo/– mutant embryos demonstrates that NCCs are specified and migrate, but undergo cell death in areas both adjacent and distal to where Fgf8 is normally expressed. This study defines the cardiovascular defects present in Fgf8 mutants and supports a role for Fgf8 in development of all the pharyngeal arches and in NCC survival.

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Doc2b Ca2+ binding site mutants enhance synaptic release at rest at the expense of sustained synaptic strength

Scientific Reports ◽

10.1038/s41598-019-50684-1 ◽

2019 ◽

Vol 9 (1) ◽

Author(s):

Quentin Bourgeois-Jaarsma ◽

Matthijs Verhage ◽

Alexander J. Groffen

Keyword(s):

Binding Site ◽

Neurotransmitter Release ◽

Action Potentials ◽

Synaptic Strength ◽

Cultured Neurons ◽

Loss Of Function ◽

Short Term ◽

Synaptic Release ◽

Activated State ◽

Primary Cultured Neurons

Abstract Communication between neurons involves presynaptic neurotransmitter release which can be evoked by action potentials or occur spontaneously as a result of stochastic vesicle fusion. The Ca2+-binding double C2 proteins Doc2a and –b were implicated in spontaneous and asynchronous evoked release, but the mechanism remains unclear. Here, we compared wildtype Doc2b with two Ca2+ binding site mutants named DN and 6A, previously classified as gain- and loss-of-function mutants. They carry the substitutions D218,220N or D163,218,220,303,357,359A respectively. We found that both mutants bound phospholipids at low Ca2+ concentrations and were membrane-associated in resting neurons, thus mimicking a Ca2+-activated state. Their overexpression in hippocampal primary cultured neurons had similar effects on spontaneous and evoked release, inducing high mEPSC frequencies and increased short-term depression. Together, these data suggest that the DN and 6A mutants both act as gain-of-function mutants at resting conditions.

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