scholarly journals Small Molecule Screening in Zebrafish Embryos Identifies Signaling Pathways Regulating Early Thyroid Development

2019 ◽  
Author(s):  
Benoit Haerlingen ◽  
Robert Opitz ◽  
Isabelle Vandernoot ◽  
Achim Trubiroha ◽  
Pierre Gillotay ◽  
...  
Keyword(s):  
Signaling Pathways ◽  
Small Molecule ◽  
Molecular Mechanisms ◽  
Model Organisms ◽  
Thyroid Cell ◽  
Zebrafish Embryos ◽  
Cardiovascular Development ◽  
Drug Induced ◽  
Thyroid Development

AbstractBackgroundDefects in embryonic development of the thyroid gland are a major cause for congenital hypothyroidism in human newborns but the underlying molecular mechanisms are still poorly understood. Organ development relies on a tightly regulated interplay between extrinsic signaling cues and cell intrinsic factors. At present, however, there is limited knowledge about the specific extrinsic signaling cues that regulate foregut endoderm patterning, thyroid cell specification and subsequent morphogenetic processes in thyroid development.MethodsTo begin to address this problem in a systematic way, we used zebrafish embryos to perform a series of in vivo phenotype-driven chemical genetic screens to identify signaling cues regulating early thyroid development. For this purpose, we treated zebrafish embryos during different developmental periods with a panel of small molecule compounds known to manipulate the activity of major signaling pathways and scored phenotypic deviations in thyroid, endoderm and cardiovascular development using whole mount in situ hybridization and transgenic fluorescent reporter models.ResultsSystematic assessment of drugged embryos recovered a range of thyroid phenotypes including expansion, reduction or lack of the early thyroid anlage, defective thyroid budding as well as hypoplastic, enlarged or overtly disorganized presentation of the thyroid primordium after budding. Our pharmacological screening identified BMP and FGF signaling as key factors for thyroid specification and early thyroid organogenesis, highlight the importance of low Wnt activities during early development for thyroid specification and implicate drug-induced cardiac and vascular anomalies as likely indirect mechanisms causing various forms of thyroid dysgenesis.ConclusionsBy integrating the outcome of our screening efforts with previously available information from other model organisms including Xenopus, chicken and mouse, we conclude that signaling cues regulating thyroid development appear broadly conserved across vertebrates. We therefore expect that observations made in zebrafish can inform mammalian models of thyroid organogenesis to further our understanding of the molecular mechanisms of congenital thyroid diseases.

scholarly journals Upregulation of GBP1 in thyroid primordium is required for developmental thyroid morphogenesis

2021 ◽  
Author(s):  
Rui-Meng Yang ◽  
Ming Zhan ◽  
Qin-Yi Zhou ◽  
Xiao-Ping Ye ◽  
Feng-Yao Wu ◽  
...  
Keyword(s):  
Congenital Hypothyroidism ◽  
Messenger Rna ◽  
Molecular Mechanisms ◽  
Cellular Adhesion ◽  
Model Organisms ◽  
Growth Defect ◽  
Zebrafish Embryos ◽  
Zebrafish Model ◽  
Thyroid Cells ◽  
Thyroid Development

Abstract Purpose Congenital hypothyroidism (CH) is a common congenital endocrine disorder in humans. CH-related diseases such as athyreosis, thyroid ectopy, and hypoplasia are primarily caused by dysgenic thyroid development. However, the underlying molecular mechanisms remain unknown. Methods To identify novel CH candidate genes, 192 CH patients were enrolled, and target sequencing of 21 known CH-related genes was performed. The remaining 98 CH patients carrying no known genes were subjected to exome sequencing (ES). The functions of the identified variants were confirmed using thyroid epithelial cells in vitro and in zebrafish model organisms in vivo. Results Four pathogenic GBP1 variations from three patients were identified. In zebrafish embryos, gbp1 knockdown caused defective thyroid primordium morphogenesis and hypothyroidism. The thyroid cells were stuck together and failed to dissociate from each other to form individual follicles in gbp1-deficient embryos. Furthermore, defects were restored with wild-type human GBP1 (hGBP1) messenger RNA (mRNA) except for mutated hGBP1 (p.H150Y, p.L187P) overexpression. GBP1 promoted β-catenin translocation into the cytosol and suppressed the formation of cellular adhesion complexes. Suppression of cell–cell adhesion restored the thyroid primordium growth defect observed in gbp1-deficient zebrafish embryos. Conclusion This study provides further understanding regarding thyroid development and shows that defective cellular remodeling could cause congenital hypothyroidism.

scholarly journals Enhanced canonical Wnt signaling during early zebrafish development perturbs the interaction of cardiac mesoderm and pharyngeal endoderm and causes thyroid specification defects

2019 ◽  
Author(s):  
Isabelle Vandernoot ◽  
Benoît Haerlingen ◽  
Achim Trubiroha ◽  
Pierre Gillotay ◽  
Véronique Janssens ◽  
...  
Keyword(s):  
Wnt Signaling ◽  
Molecular Mechanisms ◽  
Cardiac Development ◽  
Wnt Signaling Pathway ◽  
Bmp Signaling ◽  
Thyroid Cell ◽  
Zebrafish Embryos ◽  
Cardiovascular Development ◽  
Fluorescent Reporter ◽  
Cardiac Mesoderm

AbstractBackgroundCongenital hypothyroidism (CH) due to thyroid dysgenesis is a frequent congenital endocrine disorder for which the molecular mechanisms remain unresolved in the far majority of cases. This situation reflects in part our still limited knowledge about the mechanisms involved in the early steps of thyroid specification from the endoderm, in particular the extrinsic signaling cues that regulate foregut endoderm patterning. In this study, we used small molecules and genetic zebrafish models to characterize the role of various signaling pathways in thyroid specification.MethodsWe treated zebrafish embryos during different developmental periods with small molecule compounds known to modulate the activity of Wnt signaling pathway and observed effects in thyroid, endoderm and cardiovascular development using whole mount in situ hybridization and transgenic fluorescent reporter models. We used an antisense morpholino to create a zebrafish acardiac model. For thyroid rescue experiments, BMP pathway induction in zebrafish embryos was obtained by using heatshock inducible transgenic lines.ResultsInterestingly, combined analyses of thyroid and cardiovascular development revealed that overactivation of Wnt signaling during early development leads to impaired thyroid specification concurrent with severe defects in the cardiac specification. When using a model of morpholino-induced blockage of cardiomyocyte differentiation, a similar correlation was observed, suggesting that defective signaling between cardiac mesoderm and endodermal thyroid precursors contributes to thyroid specification impairment. Rescue experiments through transient overactivation of BMP signaling could partially restore thyroid specification in models with defective cardiac development.ConclusionCollectively, our results indicate that BMP signaling is critically required for thyroid cell specification and identify cardiac mesoderm as a likely source of BMP signals.

scholarly journals Small-Molecule Screening in Zebrafish Embryos Identifies Signaling Pathways Regulating Early Thyroid Development

Thyroid ◽  
2019 ◽  
Vol 29 (11) ◽  
pp. 1683-1703 ◽  
Author(s):  
Benoit Haerlingen ◽  
Robert Opitz ◽  
Isabelle Vandernoot ◽  
Achim Trubiroha ◽  
Pierre Gillotay ◽  
...  
Keyword(s):  
Signaling Pathways ◽  
Small Molecule ◽  
Zebrafish Embryos ◽  
Small Molecule Screening ◽  
Thyroid Development

scholarly journals Live tracking of inter-organ communication by endogenous exosomes in vivo

2018 ◽  
Author(s):  
Frederik J Verweij ◽  
Celine Revenu ◽  
Guillaume Arras ◽  
Florent Dingli ◽  
Damarys Loew ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Extracellular Vesicles ◽  
Molecular Mechanisms ◽  
Scavenger Receptor ◽  
Model Organisms ◽  
Zebrafish Embryos ◽  
List Type ◽  
Dependent Manner ◽  
Fluorescent Reporter

SUMMARYExtracellular vesicles (EVs) are released by most cell types but the definitive demonstration of their functional relevance remains challenging due to the lack of appropriate model organisms. Here we developed anin vivomodel to study EV physiology by expressing CD63-pHluorin in zebrafish embryos. A combination of microscopy techniques and proteomic analysis allowed us to study the biogenesis, composition, transfer, uptake and fate of individual endogenous EVsin vivo. We identified an exosome population released in a syntenin-dependent manner from the Yolk Syncytial Layer into the blood circulation. These exosomes were specifically captured, endocytosed and degraded by patrolling macrophages and endothelial cells in the Caudal Vein Plexus (CVP) in a scavenger receptor and dynamin-dependent manner. Interference with exosome secretion affected CVP growth, supporting their trophic role. Altogether, our work provides a unique model to track in vivo inter-organ communication by endogenous exosomes at individual vesicle level and high spatio-temporal accuracy.Highlights- Single endogenous EVs can be live-visualized in the whole embryo with CD63-pHluorin- In the YSL, syntenin regulates exosome release into the blood for their propagation- YSL exosomes reach the tail to be taken up by macrophages and endothelial cells- Uptake is scavenger receptor and dynamin-dependent and provides trophic supportBlurbWe propose zebrafish embryos expressing a fluorescent reporter for exosomes as a relevant model organism to live-track production, journey and fate of individual extracellular vesicles in vivo. Our model allows investigation of the composition of EVs and the molecular mechanisms controlling their biogenesis and fate and functions in receiving cells.

scholarly journals Proanthocyanidins against Oxidative Stress: From Molecular Mechanisms to Clinical Applications

2018 ◽  
Vol 2018 ◽  
pp. 1-11 ◽  
Author(s):  
Lingyu Yang ◽  
Dehai Xian ◽  
Xia Xiong ◽  
Rui Lai ◽  
Jing Song ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Signaling Pathways ◽  
Molecular Mechanisms ◽  
Metabolic Diseases ◽  
Low Cost ◽  
Natural Agents ◽  
Molecular Damage ◽  
And Control

Proanthocyanidins (PCs) are naturally occurring polyphenolic compounds abundant in many vegetables, plant skins (rind/bark), seeds, flowers, fruits, and nuts. Numerousin vitroandin vivostudies have demonstrated myriad effects potentially beneficial to human health, such as antioxidation, anti-inflammation, immunomodulation, DNA repair, and antitumor activity. Accumulation of prooxidants such as reactive oxygen species (ROS) exceeding cellular antioxidant capacity results in oxidative stress (OS), which can damage macromolecules (DNA, lipids, and proteins), organelles (membranes and mitochondria), and whole tissues. OS is implicated in the pathogenesis and exacerbation of many cardiovascular, neurodegenerative, dermatological, and metabolic diseases, both through direct molecular damage and secondary activation of stress-associated signaling pathways. PCs are promising natural agents to safely prevent acute damage and control chronic diseases at relatively low cost. In this review, we summarize the molecules and signaling pathways involved in OS and the corresponding therapeutic mechanisms of PCs.

scholarly journals High-resolution cardiovascular function confirms functional orthology of myocardial contractility pathways in zebrafish

2010 ◽  
Vol 42 (2) ◽  
pp. 300-309 ◽  
Author(s):  
Jordan T. Shin ◽  
Eugene V. Pomerantsev ◽  
John D. Mably ◽  
Calum A. MacRae
Keyword(s):  
High Speed ◽  
Dynamic Range ◽  
Model Organism ◽  
Cardiovascular Physiology ◽  
Zebrafish Embryos ◽  
Cardiovascular Development ◽  
Zebrafish Model ◽  
Ventricular Performance ◽  
Pharmacological Agents

Phenotype-driven screens in larval zebrafish have transformed our understanding of the molecular basis of cardiovascular development. Screens to define the genetic determinants of physiological phenotypes have been slow to materialize as a result of the limited number of validated in vivo assays with relevant dynamic range. To enable rigorous assessment of cardiovascular physiology in living zebrafish embryos, we developed a suite of software tools for the analysis of high-speed video microscopic images and validated these, using established cardiomyopathy models in zebrafish as well as modulation of the nitric oxide (NO) pathway. Quantitative analysis in wild-type fish exposed to NO or in a zebrafish model of dilated cardiomyopathy demonstrated that these tools detect significant differences in ventricular chamber size, ventricular performance, and aortic flow velocity in zebrafish embryos across a large dynamic range. These methods also were able to establish the effects of the classic pharmacological agents isoproterenol, ouabain, and verapamil on cardiovascular physiology in zebrafish embryos. Sequence conservation between zebrafish and mammals of key amino acids in the pharmacological targets of these agents correlated with the functional orthology of the physiological response. These data provide evidence that the quantitative evaluation of subtle physiological differences in zebrafish can be accomplished at a resolution and with a dynamic range comparable to those achieved in mammals and provides a mechanism for genetic and small-molecule dissection of functional pathways in this model organism.

scholarly journals Heavily Armed Ancestors: CRISPR Immunity and Applications in Archaea with a Comparative Analysis of CRISPR Types in Sulfolobales

Biomolecules ◽  
2020 ◽  
Vol 10 (11) ◽  
pp. 1523
Author(s):  
Isabelle Anna Zink ◽  
Erika Wimmer ◽  
Christa Schleper
Keyword(s):  
Comparative Analysis ◽  
Molecular Mechanisms ◽  
Model Organisms ◽  
Special Focus ◽  
Natural Environments ◽  
Type I ◽  
Accessory Proteins ◽  
Type Iii

Prokaryotes are constantly coping with attacks by viruses in their natural environments and therefore have evolved an impressive array of defense systems. Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) is an adaptive immune system found in the majority of archaea and about half of bacteria which stores pieces of infecting viral DNA as spacers in genomic CRISPR arrays to reuse them for specific virus destruction upon a second wave of infection. In detail, small CRISPR RNAs (crRNAs) are transcribed from CRISPR arrays and incorporated into type-specific CRISPR effector complexes which further degrade foreign nucleic acids complementary to the crRNA. This review gives an overview of CRISPR immunity to newcomers in the field and an update on CRISPR literature in archaea by comparing the functional mechanisms and abundances of the diverse CRISPR types. A bigger fraction is dedicated to the versatile and prevalent CRISPR type III systems, as tremendous progress has been made recently using archaeal models in discerning the controlled molecular mechanisms of their unique tripartite mode of action including RNA interference, DNA interference and the unique cyclic-oligoadenylate signaling that induces promiscuous RNA shredding by CARF-domain ribonucleases. The second half of the review spotlights CRISPR in archaea outlining seminal in vivo and in vitro studies in model organisms of the euryarchaeal and crenarchaeal phyla, including the application of CRISPR-Cas for genome editing and gene silencing. In the last section, a special focus is laid on members of the crenarchaeal hyperthermophilic order Sulfolobales by presenting a thorough comparative analysis about the distribution and abundance of CRISPR-Cas systems, including arrays and spacers as well as CRISPR-accessory proteins in all 53 genomes available to date. Interestingly, we find that CRISPR type III and the DNA-degrading CRISPR type I complexes co-exist in more than two thirds of these genomes. Furthermore, we identified ring nuclease candidates in all but two genomes and found that they generally co-exist with the above-mentioned CARF domain ribonucleases Csx1/Csm6. These observations, together with published literature allowed us to draft a working model of how CRISPR-Cas systems and accessory proteins cross talk to establish native CRISPR anti-virus immunity in a Sulfolobales cell.

scholarly journals Antioxidant, Anti-Inflammatory and Anti-Angiogenic Properties of Citrus lumia Juice

2020 ◽  
Vol 11 ◽  
Author(s):  
Antonella Smeriglio ◽  
Marcella Denaro ◽  
Valeria D’Angelo ◽  
Maria Paola Germanò ◽  
Domenico Trombetta
Keyword(s):  
Ascorbic Acid ◽  
Acid Content ◽  
Molecular Mechanisms ◽  
Biological Effects ◽  
Ascorbic Acid Content ◽  
Zebrafish Embryos ◽  
Anti Inflammatory ◽  
Dose Dependent

Citrus juices are a rich source of bioactive compounds with various and well-known health benefits. The aim of this study was to investigate the polyphenols and ascorbic acid content as well as to investigate the antioxidant, anti-inflammatory and anti-angiogenic properties of the juice of an ancient Mediterranean species, Citrus lumia Risso (CLJ). The antioxidant and anti-inflammatory activities were evaluated by several in vitro cell-free and cell-based assays, whereas two different in vivo models, the chick chorioallantoic membrane (CAM) and the zebrafish embryos, were used to characterize the anti-angiogenic properties. Twenty-eight polyphenols were identified by RP-LC-DAD-ESI-MS analysis (flavonoids 68.82% and phenolic acids 31.18%) with 1-caffeoyl-5-feruloylquinic acid and kaempferol 3′-rhamnoside, which represent the most abundant compounds (25.70 and 23.12%, respectively). HPLC-DAD analysis showed a high ascorbic acid content (352 mg/kg of CLJ), which contributes with polyphenols to the marked and dose-dependent antioxidant and anti-inflammatory properties observed. CLJ showed strong and dose-dependent anti-angiogenic activity as highlighted by the inhibition of blood vessel formation on CAMs and the decrease of endogenous alkaline phosphatase on zebrafish embryos. Moreover, within the concentration range tested, no dead or malformed embryos were recorded. Certainly, further studies are needed to investigate the molecular mechanisms underlying these promising biological effects, but considering the evidence of the present study, the use of CLJ as a ready-to drink safe prevention strategy for inflammatory-based diseases correlated to angiogenesis could be justified.

scholarly journals Cessation of contraction induces cardiomyocyte remodeling during zebrafish cardiogenesis

2014 ◽  
Vol 306 (3) ◽  
pp. H382-H395 ◽  
Author(s):  
Jingchun Yang ◽  
Katherine A. Hartjes ◽  
Timothy J. Nelson ◽  
Xiaolei Xu
Keyword(s):  
Signaling Pathways ◽  
Heart Development ◽  
Myosin Ii ◽  
Cardiac Contraction ◽  
Zebrafish Embryos ◽  
Mechanical Forces ◽  
In Vivo Animal Model ◽  
Lateral Fusion ◽  
Phosphoinositide 3 Kinase

Contraction regulates heart development via a complex mechanotransduction process controlled by various mechanical forces. Here, we exploit zebrafish embryos as an in vivo animal model to discern the contribution from different mechanical forces and identify the underlying mechanotransductive signaling pathways of cardiogenesis. We treated 2 days postfertilization zebrafish embryos with Blebbistatin, a myosin II inhibitor, to stop cardiac contraction, which induces a response termed cessation of contraction-induced cardiomyocyte (CM) enlargement (CCE). Accompanying the CCE, lateral fusion of myofibrils was attenuated within CMs. The CCE can be blunted by loss of blood in tail-docked zebrafish but not in cloche mutant fish, suggesting that transmural pressure rather than shear stress is accountable for the chamber enlargement. By screening a panel of small molecule inhibitors, our data suggested essential functions of phosphoinositide 3-kinase signaling and protein synthesis in CCE, which are independent of the sarcomere integrity. In summary, we defined a unique CCE response in genetically tractable zebrafish embryos. A panel of assays was established to verify the contribution from extrinsic forces and interrogate underlying signaling pathways.

scholarly journals Targeting TRIM54/Axin1/β-Catenin Axis Prohibits Proliferation and Metastasis in Hepatocellular Carcinoma

2021 ◽  
Vol 11 ◽  
Author(s):  
Jinrong Zhu ◽  
Yongqi Wu ◽  
Shaoxi Lao ◽  
Jianfei Shen ◽  
Yijian Yu ◽  
...  
Keyword(s):  
Hepatocellular Carcinoma ◽  
Prognostic Factor ◽  
Small Molecule ◽  
Therapeutic Target ◽  
Molecular Mechanisms ◽  
Proliferation And Metastasis ◽  
Oncogenic Gene ◽  
Sustained Activation

Accumulating evidence demonstrates that dysregulation of ubiquitin-mediated degradation of oncogene or suppressors plays an important role in several diseases. However, the function and molecular mechanisms of ubiquitin ligases underlying hepatocellular carcinoma (HCC) remain elusive. In the current study, we show that overexpression of TRIM54 was associated with HCC progression. TRIM54 overexpression facilitates proliferation and lung metastasis; however, inhibition of TRIM54 significantly suppressed HCC progression both in vitro and in vivo. Mechanically, we demonstrated that TRIM54 directly interacts with Axis inhibition proteins 1 (Axin1) and induces E3 ligase-dependent proteasomal turnover of Axin1 and substantially induces sustained activation of wnt/β-catenin in HCC cell lines. Furthermore, we showed that inhibition of the wnt/β-catenin signaling pathway via small molecule inhibitors significantly suppressed TRIM54-induced proliferation. Our data suggest that TRIM54 might function as an oncogenic gene and targeting the TRIM54/Axin1/β-catenin axis signaling may be a promising prognostic factor and a valuable therapeutic target for HCC.

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