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BioRxiv : the Preprint Server For... Nov 2023Cnidarians have become valuable models for understanding many aspects of developmental biology including the evolution of body plan diversity, novel cell type...
UNLABELLED
Cnidarians have become valuable models for understanding many aspects of developmental biology including the evolution of body plan diversity, novel cell type specification, and regeneration. Most of our understanding of gene function during early development in cnidarians comes from a small number of experimental systems including the sea anemone, . Few molecular tools have been developed for use in hard corals, limiting our understanding of this diverse and ecologically important clade. Here, we report the development of a suite of tools for manipulating and analyzing gene expression during early development in the northern star coral, . We present methods for gene knockdown using short hairpin RNAs, gene overexpression using exogenous mRNAs, and endogenous gene tagging using CRISPR-mediated gene knock-in. Combined with our ability to control spawning in the laboratory, these tools make a tractable experimental system for investigative studies of coral development. Further application of these tools will enable functional analyses of embryonic patterning and morphogenesis across Anthozoa and open new frontiers in coral biology research.
SUMMARY STATEMENT
This study reports the development of the first transgenic knock-in coral, providing the opportunity to track the behavior of various cell types during early coral development.
PubMed: 38948709
DOI: 10.1101/2023.11.16.567385 -
Journal of Cellular Biochemistry Jun 2024Calmodulin (CaM) is a ubiquitous, small cytosolic calcium (Ca)-binding sensor that plays a vital role in many cellular processes by binding and regulating the activity...
Calmodulin (CaM) is a ubiquitous, small cytosolic calcium (Ca)-binding sensor that plays a vital role in many cellular processes by binding and regulating the activity of over 300 protein targets. In cardiac muscle, CaM modulates directly or indirectly the activity of several proteins that play a key role in excitation-contraction coupling (ECC), such as ryanodine receptor type 2 (RyR2), l-type Ca (Ca1.2), sodium (NaV1.5) and potassium (KV7.1) channels. Many recent clinical and genetic studies have reported a series of CaM mutations in patients with life-threatening arrhythmogenic syndromes, such as long QT syndrome (LQTS) and catecholaminergic polymorphic ventricular tachycardia (CPVT). We recently showed that four arrhythmogenic CaM mutations (N98I, D132E, D134H, and Q136P) significantly reduce the binding of CaM to RyR2. Herein, we investigate in vivo functional effects of these CaM mutations on the normal zebrafish embryonic heart function by microinjecting complementary RNA corresponding to CaM, CaM, CaM, and CaM mutants. Expression of CaM and CaM mutants results in significant reduction of the zebrafish heart rate, mimicking a severe form of human bradycardia, whereas expression of CaM results in an increased heart rate mimicking human ventricular tachycardia. Moreover, analysis of cardiac ventricular rhythm revealed that the CaM and CaM zebrafish groups display an irregular pattern of heart beating and increased amplitude in comparison to the control groups. Furthermore, circular dichroism spectroscopy experiments using recombinant CaM proteins reveals a decreased structural stability of the four mutants compared to the wild-type CaM protein in the presence of Ca. Finally, Ca-binding studies indicates that all CaM mutations display reduced CaM Ca-binding affinities, with CaM exhibiting the most prominent change. Our data suggest that CaM mutations can trigger different arrhythmogenic phenotypes through multiple and complex molecular mechanisms.
PubMed: 38946237
DOI: 10.1002/jcb.30619 -
Journal of Proteome Research Jun 2024Tumor necrosis factor (TNF) has well-established roles in neuroinflammatory disorders, but the effect of TNF on the biochemistry of brain cells remains poorly...
Tumor necrosis factor (TNF) has well-established roles in neuroinflammatory disorders, but the effect of TNF on the biochemistry of brain cells remains poorly understood. Here, we microinjected TNF into the brain to study its impact on glial and neuronal metabolism (glycolysis, pentose phosphate pathway, citric acid cycle, pyruvate dehydrogenase, and pyruvate carboxylase pathways) using C NMR spectroscopy on brain extracts following intravenous [1,2-C]-glucose (to probe glia and neuron metabolism), [2-C]-acetate (probing astrocyte-specific metabolites), or [3-C]-lactate. An increase in [4,5-C]-glutamine and [2,3-C]-lactate coupled with a decrease in [4,5-C]-glutamate was observed in the [1,2-C]-glucose-infused animals treated with TNF. As glutamine is produced from glutamate by astrocyte-specific glutamine synthetase the increase in [4,5-C]-glutamine reflects increased production of glutamine by astrocytes. This was confirmed by infusion with astrocyte substrate [2-C]-acetate. As lactate is metabolized in the brain to produce glutamate, the simultaneous increase in [2,3-C]-lactate and decrease in [4,5-C]-glutamate suggests decreased lactate utilization, which was confirmed using [3-C]-lactate as a metabolic precursor. These results suggest that TNF rearranges the metabolic network, disrupting the energy supply chain perturbing the glutamine-glutamate shuttle between astrocytes and the neurons. These insights pave the way for developing astrocyte-targeted therapeutic strategies aimed at modulating effects of TNF to restore metabolic homeostasis in neuroinflammatory disorders.
PubMed: 38943617
DOI: 10.1021/acs.jproteome.4c00035 -
Journal of Neurovirology Jun 2024The Rabies virus is a neurotropic virus that manipulates the natural cell death processes of its host to ensure its own survival and replication. Studies have shown that...
The Rabies virus is a neurotropic virus that manipulates the natural cell death processes of its host to ensure its own survival and replication. Studies have shown that the anti-apoptotic effect of the virus is mediated by one of its protein named, rabies glycoprotein (RVG). Alzheimer's disease (AD) is characterized by the loss of neural cells and memory impairment. We aim to examine whether expression of RVG in the hippocampal cells can shield the detrimental effects induced by Aβ. Oligomeric form of Aβ (oAβ) or vehicle was bilaterally microinjected into the dorsal hippocampus of male Wistar rats. One week later, two μl (10 T.U. /ml) of the lentiviral vector carrying RVG gene was injected into their dorsal hippocampus (post-treatment). In another experiment, the lentiviral vector was microinjected one week before Aβ injection (pre-treatment). One week later, the rat's brain was sliced into cross-sections, and the presence of RVG-expressing neuronal cells was confirmed using fluorescent microscopy. Rats were subjected to assessments of spatial learning and memory as well as passive avoidance using the Morris water maze (MWM) and the Shuttle box apparatuses, respectively. Protein expression of AMPA receptor subunit (GluA1) was determined using western blotting technique. In MWM, Aβ treated rats showed decelerated acquisition of the task and impairment of reference memory. RVG expression in the hippocampus prevented and restored the deficits in both pre- and post- treatment conditions, respectively. It also improved inhibitory memory in the oAβ treated rats. RVG increased the expression level of GluA1 level in the hippocampus. Based on our findings, the expression of RVG in the hippocampus has the potential to enhance both inhibitory and spatial learning abilities, ultimately improving memory performance in an AD rat model. This beneficial effect is likely attributed, at least in part, to the increased expression of GluA1-containing AMPA receptors.
PubMed: 38943023
DOI: 10.1007/s13365-024-01221-y -
STAR Protocols Jun 2024A gene-rescue experiment under a mutant background is essential to clarify gene function and the resulting biological potential in vivo. Here, we present a protocol for...
A gene-rescue experiment under a mutant background is essential to clarify gene function and the resulting biological potential in vivo. Here, we present a protocol for determining the change in interferon response by microinjecting plasmids into one-cell-stage zebrafish embryos. We describe steps for comparing the resistance potential to virus infection in wild-type and knockout zebrafish larvae following plasmid microinjection. We then detail how to link the enhanced interferon immunity to the improved resistance in knockout zebrafish larvae by gene-rescue experiments. For complete details on the use and execution of this protocol, please refer to Qu et al..
PubMed: 38941183
DOI: 10.1016/j.xpro.2024.103156 -
Free Radical Biology & Medicine Jun 2024Disturbance in iron homeostasis has been described in Parkinson's disease (PD), in which iron regulatory protein 2 (IRP2) plays a crucial role. IRP2 deletion resulted in...
Disturbance in iron homeostasis has been described in Parkinson's disease (PD), in which iron regulatory protein 2 (IRP2) plays a crucial role. IRP2 deletion resulted in the misregulation of iron metabolism and subsequent neurodegeneration. However, growing evidence showed that the levels of IRP2 were increased in the substantial nigra (SN) in MPTP-induced PD mice. To further clarify the role of increased IRP2 in PD, we developed IRP2-overexpressed mice by microinjecting AAV-Ireb2 in the SN. These mice showed decreased motor ability, abnormal gait and anxiety. Iron deposits induced by increased TFR1 and dopaminergic neuronal loss were observed in the SN. When these mice were treated with MPTP, exacerbated dyskinesia and dopaminergic neuronal loss were observed. In addition, TP53 was post-transcriptionally upregulated by IRP2 binding to the iron regulated element (IRE) in its 3' untranslated region. This resulted in increased lipid peroxidation levels and induced ferroptosis through the SLC7A11-ALOX12 pathway, which was independent of GPX4. This study revealed that IRP2 homeostasis in the SN was critical for PD progression and clarified the molecular mechanism of ferroptosis caused by IRP2.
PubMed: 38936518
DOI: 10.1016/j.freeradbiomed.2024.06.020 -
Reproductive Biology Jun 2024In mammals, early embryogenesis relies heavily on the regulation of maternal transcripts including protein-coding and non-coding RNAs stored in oocytes. In this study,...
In mammals, early embryogenesis relies heavily on the regulation of maternal transcripts including protein-coding and non-coding RNAs stored in oocytes. In this study, the expression of three bovine oocyte expressed long non-coding RNAs (lncRNAs), OOSNCR1, OOSNCR2, and OOSNCR3, was characterized in somatic tissues, the ovarian follicle, and throughout early embryonic development. Moreover, the functional requirement of each transcript during oocyte maturation and early embryonic development was investigated using a siRNA-mediated knockdown approach. Tissue distribution analysis revealed that OOSNCR1, OOSNCR2 and OOSNCR3 are predominantly expressed in fetal ovaries. Follicular cell expression analysis revealed that these lncRNAs are highly expressed in the oocytes, with minor expression detected in the cumulus cells (CCs) and mural granulosa cells (mGCs). The expression for all three genes was highest during oocyte maturation, decreased at fertilization, and ceased altogether by the 16-cell stage. Knockdown of OOSNCR1, OOSNCR2 and OOSNCR3 in immature oocytes was achieved by microinjection of the cumulus-enclosed germinal vesicle (GV) oocytes with siRNAs targeting these lncRNAs. Knockdown of OOSNCR1, OOSNCR2 and OOSNCR3 did not affect cumulus expansion, but oocyte survival at 12 h post-insemination was significantly reduced. In addition, knockdown of OOSNCR1, OOSNCR2 and OOSNCR3 in immature oocytes resulted in a decreased rate of blastocyst development, and reduced expression of genes associated with oocyte competency such as nucleoplasmin 2 (NPM2), growth differentiation factor 9 (GDF9), bone morphogenetic protein 15 (BMP15), and JY-1 in MII oocytes. The data herein suggest a functional requirement of OOSNCR1, OOSNCR2, and OOSNCR3 during bovine oocyte maturation and early embryogenesis.
PubMed: 38936296
DOI: 10.1016/j.repbio.2024.100915 -
International Journal of Molecular... Jun 2024A large diversity of epigenetic factors, such as microRNAs and histones modifications, are known to be capable of regulating gene expression without altering DNA...
A large diversity of epigenetic factors, such as microRNAs and histones modifications, are known to be capable of regulating gene expression without altering DNA sequence itself. In particular, miR-1 is considered the first essential microRNA in cardiac development. In this study, miR-1 potential role in early cardiac chamber differentiation was analyzed through specific signaling pathways. For this, we performed in chick embryos functional experiments by means of miR-1 microinjections into the posterior cardiac precursors-of both primitive endocardial tubes-committed to sinoatrial region fates. Subsequently, embryos were subjected to whole mount in situ hybridization, immunohistochemistry and RT-qPCR analysis. As a relevant novelty, our results revealed that miR-1 increased , and , while this microRNA diminished and expressions during early differentiation of the cardiac sinoatrial region. Furthermore, we observed in this developmental context that miR-1 upregulated and and downregulated , which are three crucial factors in the retinoic acid signaling pathway. Interestingly, we also noticed that miR-1 directly interacted with and /, as well as with /, which are three key factors actively involved in regulation. Our study shows, for the first time, a key role of miR-1 as an epigenetic regulator in the early differentiation of the cardiac sinoatrial region through orchestrating opposite actions between retinoic acid and , fundamental to properly assign cardiac cells to their respective heart chambers. A better understanding of those molecular mechanisms modulated by miR-1 will definitely help in fields applied to therapy and cardiac regeneration and repair.
Topics: Animals; MicroRNAs; Epigenesis, Genetic; Cell Differentiation; Gene Expression Regulation, Developmental; Chick Embryo; MEF2 Transcription Factors; Sinoatrial Node; Signal Transduction; Heart
PubMed: 38928314
DOI: 10.3390/ijms25126608 -
International Journal of Molecular... Jun 2024Aneurysms pose life-threatening risks due to the dilatation of the arteries and carry a high risk of rupture. Despite continuous research efforts, there are still no...
Aneurysms pose life-threatening risks due to the dilatation of the arteries and carry a high risk of rupture. Despite continuous research efforts, there are still no satisfactory or clinically effective pharmaceutical treatments for this condition. Accelerated inflammatory processes during aneurysm development lead to increased levels of matrix metalloproteinases (MMPs) and destabilization of the vessel wall through the degradation of the structural components of the extracellular matrix (ECM), mainly collagen and elastin. Tissue inhibitors of metalloproteinases (TIMPs) directly regulate MMP activity and consequently inhibit ECM proteolysis. In this work, the synthesis of TIMP-1 protein was increased by the exogenous delivery of synthetic TIMP-1 encoding mRNA into aortic vessel tissue in an attempt to inhibit MMP-9. In vitro, TIMP-1 mRNA transfection resulted in significantly increased TIMP-1 protein expression in various cells. The functionality of the expressed protein was evaluated in an appropriate ex vivo aortic vessel model. Decreased MMP-9 activity was detected using in situ zymography 24 h and 48 h post microinjection of 5 µg TIMP-1 mRNA into the aortic vessel wall. These results suggest that TIMP-1 mRNA administration is a promising approach for the treatment of aneurysms.
Topics: Tissue Inhibitor of Metalloproteinase-1; Matrix Metalloproteinase 9; RNA, Messenger; Animals; Humans; Rats; Aneurysm; Aorta; Male; Arteries; Matrix Metalloproteinase Inhibitors
PubMed: 38928311
DOI: 10.3390/ijms25126599 -
Environmental Toxicology and Chemistry Jun 2024Benzotriazole ultraviolet stabilizers (BUVSs) are a group of widely used chemicals added to a variety of consumer (e.g., plastics) and industrial (e.g., metal coating)...
Benzotriazole ultraviolet stabilizers (BUVSs) are a group of widely used chemicals added to a variety of consumer (e.g., plastics) and industrial (e.g., metal coating) goods. Although detected globally as an environmentally persistent pollutant, BUVSs have received relatively little toxicological attention and only recently have been acknowledged to affect development and the endocrine system in vivo. In our previous study, altered behavior, indicative of potential neurotoxicity, was observed among rainbow trout alevins (day 14 posthatching) that were microinjected as embryos with a single environmentally relevant dose of 2,4-di-tert-butyl-6-(5-chloro-2H-benzotriazol-2-yl) phenol (UV-327). In the present follow-up study, we performed whole-transcriptome profiling (RNA sequencing) of newly hatched alevins from the same batch. The primary aim was to identify biomarkers related to behavior and neurology. Dose-specifically, 1 to 176 differentially expressed genes (DEGs) were identified. In the group presenting altered behavior (273.4 ng g), 176 DEGs were identified, yet only a fraction was related to neurological functions, including water, calcium, and potassium homeostasis; acetylcholine transmission and signaling; as well insulin and energy metabolism. The second objective was to estimate the transcriptomic point of departure (tPOD) and assess if point estimate(s) are protective of altered behavior. A tPOD was established at 35 to 94 ng UV-327 g egg, making this tPOD protective of behavioral alterations. Holistically, these transcriptomic alterations provide a foundation for future research on how BUVSs can influence rainbow trout alevin development, while providing support to the hypothesis that UV-327 can influence neurogenesis and subsequent behavioral endpoints. The exact structural and functional changes caused by embryonic exposure to UV-327 remain enigmatic and will require extensive investigation before being deciphered and understood toxicologically. Environ Toxicol Chem 2024;00:1-12. © 2024 The Author(s). Environmental Toxicology and Chemistry published by Wiley Periodicals LLC on behalf of SETAC.
PubMed: 38923588
DOI: 10.1002/etc.5943