Embryo Model Research Articles

Asiatic acid in Centella asiatica extract towards morphological development in an intermittent hypoxia intrauterine embryo model and molecular prediction pathway of insulin-like growth factor-1 receptor signalling.

Hypoxia during pregnancy generates oxidative stress that alters the growth and development of the human fetus. Insulin-like growth factor-1 (IGF-1) receptors are essential for normal fetal growth. Asiatic acid in Centella asiatica (CA) has antioxidant properties to prevent growth impairment in hypoxia. This study aimed to investigate the effect of asiatic acid on the morphological development of an intermittent hypoxia (IH) zebrafish embryo model and analyze molecular docking prediction in IGF-1 receptor (IGF-1R) signaling. Embryos of zebrafish at 2 hours postfertilization (hpf) were assigned to control negative (C), IH, and combination IH and CA extract groups consisting of 1.25 (IHCA1), 2.5 (IHCA2), and 5 (IHCA3) µg/ml. Hypoxia treatment (conducted 4 hours/day) and CA extract were administered for 3 days (2-72 hpf). The parameters of body length and head length were evaluated at 3, 6, and 9 days postfertilization (dpf). The data were analyzed by a two-way analysis of variance (p < 0.05). Molecular docking was performed to explore the binding affinity of asiatic acid to IGF-1R by Molegro Virtual Docker ver.5 software. The body length and head length of embryos in the IH and treatment groups (IHCA) were shorter than those in the control group at 3 dpf (p < 0.05). However, the body length was more prolonged in the IHCA1 group, but the head length was longer in the IHCA2 group than in the IH group at 6 and 9 dpf. Molecular docking showed the reliable interaction of asiatic acid with IGF-1R signaling in an IH animal model. The administration of CA extract benefits IH through the development and growth of zebrafish embryos at a dose of 2.5-5 µg/ml. Asiatic acid has a binding affinity for IGF-1R signaling.

Behind the developing brains and beating hearts of stem cell-derived embryo models.

Studies over the past decade have shown how stem cells representing embryonic and extra-embryonic tissues of the mouse can self-assemble in the culture dish to recapitulate an astonishing part of early embryonic development. A systematic analysis has demonstrated how pluripotent embryonic stem cells can be induced to behave like the implanting epiblast; how they can interact with trophectoderm stem cells to form a patterned structure resembling the implanting embryo prior to gastrulation; and how the third stem cell type-extra-embryonic endoderm cells-can be incorporated to generate structures that undergo the cell movements and gene expression patterns of gastrulation. Moreover, such stem cell-derived embryo models can proceed to neurulation and establish progenitors for all parts of the brain and neural tube, somites, beating heart structures and gut tube. They develop within extra-embryonic yolk sacs that initiate haematopoiesis. Here we trace this journey of discovery.

Silver nitroprusside nanoparticles for breast cancer therapy: in vitro and in vivo approach.

The advancement of nanotechnology has led to the experimental development of cancer therapeutics, which may overcome the shortcomings of commercially available drugs and facilitate improved clinical outcomes. Recently, several metal nanoparticles, especially silver, have been evaluated by scientists globally as useful chemotherapeutic agents due to their multi-functionality and well-recognized biological activity. Herein, we developed silver nitroprusside nanoparticles (abbreviated as AgNNPs) with slight modifications in the reaction conditions and demonstrated their application for breast cancer therapy using in vitro assays and in vivo experiments in a mouse model. Initially, the modified AgNNPs were thoroughly characterized using several analytical techniques. AgNNPs were found to be biocompatible according to in vitro experiments in normal cell lines (HEK-293 and EA.hy926), which was further validated by a hemolysis assay (ex vivo experiment) using mouse red blood cells. In contrast, the cell viability assay using the MTT reagent showed the cytotoxic nature of the AgNNPs against several cancer cell lines (MDA-MB-231, 4T1, B16F10, and PANC-1). Their detailed anticancer activity was investigated using 4T1 (mouse specific) and MDA-MB-231 (human specific) cells through various in vitro assays. The nanoparticles inhibited the formation of blood vessels in the chick embryo model, highlighting their anti-angiogenic behavior. Furthermore, the administration of AgNNPs significantly inhibited orthotopic breast tumor growth (4T1; BALB/c mice) and increased the survivability of the tumor-bearing mice. Also, we demonstrated the plausible molecular mechanisms for the anti-cancer activity of AgNNPs through various in vitro assays and in vivo experiments. Overall, the results support that AgNNPs can be used as an alternative generalized nanomedicine for the treatment of breast and other cancers after proper biosafety evaluation in near future.

Effect of dimenhydrinate on neural tube development in early chick embryo model

Amaç: Bu çalışmada dimenhidrinat’ın erken dönem civciv embriyo modelinde nöral tüp gelişimi üzerindeki etkilerinin araştırılması amaçlanmıştır.&#x0D; Gereç ve Yöntem: Çalışmada 32 fertil, spesifik patojen olmayan yumurta kullanıldı. Tüm yumurtalar her bir grupta 8 adet olacak şekilde dört gruba ayrıldı. İnkübasyonun 28. saatinde deney gruplarına üç farklı dozda (0,7, 1,4, 2,1 mg/kg) dimenhidrinat ve kontrol grubuna serum fizyolojik enjekte edildi. İnkübasyonun 48. saatinde tüm embriyolar diseke edildi ve ışık mikroskobu altında histolojik olarak değerlendirildi.&#x0D; Bulgular: Kontrol, düşük, orta ve yüksek doz grubu embriyoları morfolojik olarak değerlendirildiğinde, kraniokaudal uzunluk ortalamaları sırasıyla; 8493,73±1342,90 µm, 5743,61±770,3 µm, 5572,62±315,32 µm, 5306,55±1135,99 µm, somit sayıları ortalamaları sırasıyla; 15,91±1,62, 15,38±1,19, 15,13±0,83, 10,63±5,15‘dir. Dimenhidrinat dozundaki artışa bağlı olarak kraniokaudal uzunluklarının ve somit sayılarının azaldığı tespit edildi. Dimenhidrinat doz artışına bağlı olarak nöral tüp açıklığının arttığı belirlendi .&#x0D; Sonuç: Bu çalışmada, erken dönem civciv embriyo modelinde dimenhidrinat uygulanan gruplarda doz artışına bağlı olarak nörogenezisin ve morfolojik gelişimin olumsuz etkilendiği görüldü.

Self-organization principles in stem-cell-derived synthetic embryo models

As embryos, we were extremely lucky to survive implantation, when we transitioned from a simple ball of undifferentiated cells to a complex structure of distinct lineages. During development, fertilization generates a totipotent zygote that ultimately gives rise to the myriad cell types of the whole organism. In mouse embryos, the first cell fate decision leads to trophectoderm (TE) progenitors on the outside of the early embryo, surrounding the inner cell mass. Epiblast (EPI) and primitive endoderm (PE) cells then arise from the inner cell mass and are initially intermingled (embryonic day 3.5).

Toxicity Study of Chlorzoxazone and Isosorbide Dinitrate using Chick Embryo

Number of potential drugs are underutilized due to a lack of availability of teratological data. Isosorbide Dinitrate is a saviour drug in angina prophylaxis while chlorzoxazone is a skeletal muscle relaxant and there is no adequate teratogenic study performed till date. This study aims to assess the teratological effect of these drugs on vital organs using the chick embryo model. White Leghorn’s (Gallus gallus domesticus) fertilised chicken eggs were acquired from shivneri agro and hatcheries Nashik and divided into five groups (n=10) as Control, nonteratogenic, teratogenic, chlorzoxazone, and Isosorbide Dinitrate. The drug was injected via yolk inoculation and after inoculation; the eggs were re-incubated at 37.5-37.8°C and 50-60% RH for 21 days. Then the embryos were harvested and evaluated for morphological and histopathological changes. The gross macroscopic examination of Isosorbide Dinitrate and chlorzoxazone treated chicks were normal. The development of the embryo was found shunted in Isosorbide Dinitrate treated group. Microscopic abrasions found in Isosorbide Dinitrate treated group are myocardial congestion, hemorrhage, hydropic degeneration, dislocation of the nucleus, splitting of cells, and infiltration of cells at all three doses. No teratogenic response was observed in chlorzoxazone treated group hence found to be safe. Teratogenic effect of Chlorzoxazone and isosorbide dinitrate in chick embryo provided notable details. Chlorzoxazone was found to be safe in chick embryos in the developmental phase, While Isosorbide dinitrate at highest dose was found toxic and so, it is inadvisable for its utilization in pregnancy.

Chiari Malformation with Surgically Induced Open Neural Tube Defect in Late Chick Embryos : Characterization by Magnetic Resonance Imaging and Histopathological Analysis.

Chiari II malformation (CM II) is still the main cause of severe morbidity and mortality in children with open neural tube defects (ONTDs). The goal of this study was to validate a CM II model in late-stage chick embryos with surgically induced ONTDs. To make the chick embryo model of ONTD, their neural tubes were opened for a length of 5-6 somites at the thoracic level in Hamburger and Hamilton stage 18 chick embryos (n=150). They were reincubated in ovo. up to a total age of 17-21 days. A total of 19 embryos survived and were assigned to either the postoperative day (POD) 14-15 group (n=6) or the POD 17-18 group (n=13). Magnetic resonance imaging (MRI) and histopathologic findings of embryo heads with spinal ONTDs were compared with age-matched normal chick embryos. The chick embryos with ONTDs demonstrated definite and constant structural changes, such as downward displacement of the cerebellum to just above the foramen magnum and narrow and small cerebrospinal fluid spaces in the crowded small posterior fossa. These morphologic features were more prominent in the POD 17-18 group than in the POD 14-15 group. This is the first description of CM II with spinal ONTD in a late-stage chick embryo model with MRI and histopathological analysis. The morphological changes of the posterior fossa in this study mimic those of CM II associated with spinal ONTD in humans. This model will facilitate investigation of the pathogenesis of CM II.

Synthetic whole embryo models and their applications

Synthetic whole embryo models and their applications

Development of a 3D simulator for training the mouse in utero electroporation.

In utero electroporation (IUE) requires high-level training in microinjection through the mouse uterine wall into the lateral ventricle of the mouse brain. Training for IUE is currently being performed in live mice as no artificial models allow simulations yet. This study aimed to develop an anatomically realistic 3D printed simulator to train IUE in mice. To this end, we created embryo models containing lateral ventricles. We coupled them to uterus models in six steps: (1) computed tomography imaging, (2) 3D model segmentation, (3) 3D model refinement, (4) mold creation to cast the actual model, (5) 3D mold printing, and (6) mold casting the molds with a mix of soft silicones to ensure the hardness and consistency of the uterus and embryo. The results showed that the simulator assembly successfully recreated the IUE. The compression test did not differ in the mechanical properties of the real embryo or in the required load for uterus displacement. Furthermore, more than 90% of the users approved the simulator as an introduction to IUE and considered that the simulator could help reduce the number of animals for training. Despite current limitations, our 3D simulator enabled a realistic experience for initial approximations to the IUE and is a real alternative for implementing the 3Rs. We are currently working on refining the model.

Zebrafish anterior segment mesenchyme progenitors are defined by function of tfap2a but not sox10

The anterior segment is a critical component of the visual system. Developing independent of the retina, the AS relies partially on cranial neural crest cells (cNCC) as its earliest progenitors. The cNCCs are thought to first adopt a periocular mesenchyme (POM) fate and subsequently target to the AS upon formation of the rudimentary retina. AS targeted POM is termed anterior segment mesenchyme (ASM). However, it remains unknown when and how the switch from cNCC to POM or POM to ASM takes place. As such, we sought to visualize the timing of these transitions and identify the regulators of this process using the zebrafish embryo model. Using two color fluorescence in situ hybridization, we tracked cNCC and ASM target gene expression from 12 to 24hpf. In doing so, we identified a tfap2a and foxC1a co-expression at 16hpf, identifying the earliest ASM to arrive at the AS. Interestingly, expression of two other key regulators of NCC, foxD3 and sox10 was not associated with early ASM. Functional analysis of tfap2a, foxD3 and sox10 revealed that tfap2a and foxD3 are both critical regulators of ASM specification and AS formation while sox10 was dispensable for either specification or development of the AS. Using genetic knockout lines, we show that in the absence of tfap2a or foxD3 function ASM cells are not specified, and subsequently the AS is malformed. Conversely, sox10 genetic mutants or CRISPR Cas9 injected embryos displayed no defects in ASM specification, migration or the AS. Lastly, using transcriptomic analysis, we show that GFP + cNCCs derived from Tg [foxD3:GFP] and Tg [foxC1b:GFP] share expression profiles consistent with ASM development whereas cNCCs isolated from Tg [sox10:GFP] exhibit expression profiles associated with vasculogenesis, muscle function and pigmentation. Taken together, we propose that the earliest stage of anterior segment mesenchyme (ASM) specification in zebrafish is approximately 16hpf and involves tfap2a/foxC1a positive cNCCs.

Going high and low: on pluralism and neutrality in human embryology policy-making

Formulating sound and acceptable embryo research policy remains challenging especially in a pluralistic world. This challenge has acquired a new dimension of complexity with the advent of so-called embryo models,...

Cytotoxic monoaryl furazanopyrazines with microtubule destabilizing activity in the sea urchin embryo model

Biological activity of monoaryl furazanopyrazines (FPs), known as high-energy molecules, is scarcely explored. A series of monoaryl FPs was synthesized by condensation of arylglyoxals with diaminofurazane. The targeted compounds at low micromolar concentrations inhibited growth of human cancer cells in NCI60 screen. Further evaluation using a sea urchin embryo model revealed that antiproliferative activity of monoaryl FPs could be associated with targeting tubulin and microtubule destabilization, systemic (nonspecific) toxicity, or both. Introduction of hydroxy group into pyrazine ring of monoaryl FPs yielded inactive molecules.

PFAS-induced lipidomic dysregulations and their associations with developmental toxicity in zebrafish embryos

Per- and polyfluoroalkyl substances (PFASs) are persistent environmental contaminants, posing developmental toxicity to fish and human. PFAS-induced lipid metabolism disorders were demonstrated using the zebrafish (Danio rerio) embryo model, but the detailed changes of lipid compositions and the influence of these changes on the biological development are still unclear. Herein, lipidomics analysis was performed to reveal the dysregulations of lipid metabolism in zebrafish embryos exposed to perfluorooctanoic acid (PFOA) or perfluorooctane sulfonate (PFOS) through microinjection. Various abnormal phenotypes were observed, including heart bleeding, pericardium edema, spinal curvature and increased heart rate at 72 h after fertilization, especially in the PFOS exposure groups. Lipidomic profiling found downregulated phosphatidylethanolamines in the PFAS-exposed embryos, especially those containing a docosahexaenoyl (DHA) chain, indicating an excessive oxidative damage to the embryos. Glycerolipids were mainly upregulated in the PFOA groups but downregulated in the PFOS groups. These aberrations may reflect oxidative stress, energy metabolism malfunction and proinflammatory signals induced by PFASs. However, supplement of DHA may not be effective in recovering the lipidomic dysregulations and protecting from the developmental toxicity induced by PFASs, showing the complexity of the toxicological mechanisms. This work has revealed the associations between the abnormal phenotypes and dysregulations of lipid metabolism in zebrafish embryos induced by PFASs from the aspect of lipidomics, and discovered the underlying molecular mechanisms of the developmental toxicity of PFASs.

In preprints: improving and interrogating embryo models.

In preprints: improving and interrogating embryo models.

Bridging the In Vitro to In Vivo gap: Using the Chick Embryo Model to Accelerate Nanoparticle Validation and Qualification for In Vivo studies.

We postulate that nanoparticles (NPs) for use in therapeutic applications have largely not realized their clinical potential due to an overall inability to use in vitro results to predict NP performance in vivo. The avian embryo and associated chorioallantoic membrane (CAM) has emerged as an in vivo preclinical model that bridges the gap between in vitro and in vivo, enabling rapid screening of NP behavior under physiologically relevant conditions and providing a rapid, accessible, economical, and more ethical means of qualifying nanoparticles for in vivo use. The CAM is highly vascularized and mimics the diverging/converging vasculature of the liver, spleen, and lungs that serve as nanoparticle traps. Intravital imaging of fluorescently labeled NPs injected into the CAM vasculature enables immediate assessment and quantification of nano-bio interactions at the individual NP scale in any tissue of interest that is perfused with a microvasculature. In this review, we highlight how utilization of the avian embryo and its CAM as a preclinical model can be used to understand NP stability in blood and tissues, extravasation, biocompatibility, and NP distribution over time, thereby serving to identify a subset of NPs with the requisite stability and performance to introduce into rodent models and enabling the development of structure-property relationships and NP optimization without the sacrifice of large populations of mice or other rodents. We then review how the chicken embryo and CAM model systems have been used to accelerate the development of NP delivery and imaging agents by allowing direct visualization of targeted (active) and nontargeted (passive) NP binding, internalization, and cargo delivery to individual cells (of relevance for the treatment of leukemia and metastatic cancer) and cellular ensembles (e.g., cancer xenografts of interest for treatment or imaging of cancer tumors). We conclude by showcasing emerging techniques for the utilization of the CAM in future nano-bio studies.

Stem cell ethics and policy: What’s old is new again

Stem cells are increasingly being used to model human development and disease in the form of self-organizing embryo models, brain organoids, and neurological chimeras. These new research directions are resurrecting old embryo debates around moral status and personhood. Hyun considers how these old questions are tackled in these new contexts.

Treponema pallidum membrane protein Tp47 promotes angiogenesis through ROS-induced autophagy.

Pathological angiogenesis is an important manifestation of syphilis, but the underlying mechanism of Treponema pallidum subspecies pallidum (T.pallidum)-induced angiogenesis is poorly understood. The objective of this study is to investigate the role and related mechanism of the T.pallidum membrane protein Tp47 in angiogenesis. The proangiogenic activity of recombinant T.pallidum membrane protein Tp47 in human umbilical vein endothelial cells (HUVECs) was assessed by tube formation assay, three-dimensional angiogenesis analysis and experiments with a zebrafish embryo model. The effects of mitochondrial ROS and NADPH oxidase on intracellular ROS induced by Tp47 were further investigated. Furthermore, the levels of autophagy-related proteins and autophagic flux were measured. Finally, the role of ROS-induced autophagy in angiogenesis was studied. Tp47 promoted tubule formation and the formation of angiogenic sprouts in vitro. In addition, a significant increase in the number of subintestinal vessel branch points in zebrafish injected with Tp47 was observed using a zebrafish embryo model. Tp47 also significantly increased intracellular ROS levels in a dose-dependent manner. Tp47-induced tube formation and angiogenic sprout formation were effectively prevented by the ROS inhibitor NAC. In addition, Tp47 enhanced the production of mitochondrial ROS and expression of the NADPH oxidase-related proteins Nox2 and Nox4. The production of mitochondrial ROS and intracellular ROS was reduced by the NADPH oxidase inhibitors DPI and apocynin. Furthermore, Tp47 significantly increased expression of the autophagy-related proteins P62 and Beclin 1 and the LC3-II/LC3-I ratio and promoted an increase in autophagic flux, which could be effectively rescued by coincubation with the ROS inhibitor NAC. Further intervention with the autophagy inhibitor BafA1 significantly inhibited tube formation and angiogenic sprout formation. Tp47-induced NADPH oxidase enhanced intracellular ROS production via mitochondrial ROS and promoted angiogenesis through autophagy mediated by ROS. These findings may contribute to our understanding of pathological angiogenesis in syphilis.

Effects of bepridil on early cardiac development of zebrafish.

Bepridil is a commonly used medication for arrhythmia and heart failure. It primarily exerts hemodynamic effects by inhibiting Na+/K+ movement and regulating the Na+/Ca2+ exchange. In comparison to other Ca2+ inhibitors, bepridil has a long half-life and a complex pharmacology. Additionally, it is widely used in antiviral research and the treatment of various diseases. However, the toxicity of this compound and its other possible effects on embryonic development are unknown. In this study, we investigated the toxicity of bepridil on rat myocardial H9c2 cells. After treatment with bepridil, the cells became overloaded with Ca2+ and entered a state of cytoplasmic vacuolization and nuclear abnormality. Bepridil treatment resulted in several morphological abnormalities in zebrafish embryo models, including pericardium enlargement, yolk sac swelling, and growth stunting. The hemodynamic effects on fetal development resulted in abnormal cardiovascular circulation and myocardial weakness. After inhibiting the Ca2+ transmembrane, the liver of zebrafish larvae also displayed an ectopic and deficient spatial location. Additionally, the results of the RNA-seq analysis revealed the detailed gene expression profiles and metabolic responses to bepridil treatment in zebrafish embryonic development. Taken together, our study provides an important evaluation of antiarrhythmic agents for clinical use in prenatal heart patients.

Protective effects of cyanidin-3-O-glucoside on BPA-induced neurodevelopmental toxicity in zebrafish embryo model

Bisphenol A (BPA) is ubiquitous in the environment and poses a threat to wildlife and human health. It has been reported that BPA may cause the neurotoxicity during gestational and neonatal periods. Cyanidin-3-O-glucoside (C3G) is one of the most abundant anthocyanins that has shown multiple bio-functions. In this study, the protective effects and possible mechanism of C3G against BPA-induced neurodevelopment toxicity in zebrafish embryos/larvae were studied. The results showed that co-exposure of C3G (25 μg/mL) significantly attenuated BPA-induced deficit in locomotor behavior and restored the BPA-induced aberrant changes in brain morphology of zebrafish larvae. Further studies showed that the defects of central nervous development and the downregulated neurogenesis relative genes induced by BPA were significantly counteracted by co-exposure with 5 μg/mL of C3G. In addition, C3G (25 μg/mL) mitigated the decline of glutathione (GSH) content and enzymatic activities of superoxide dismutase (SOD), glutathione peroxidase (GPx) and catalase (CAT), attenuated oxidative stress and cell apoptosis induced by BPA in zebrafish. The enhancements of the expression of genes involved in the Nrf2-ARE pathway (Nrf2, HO-1, NQO1, GCLC, and GCLM) were also observed by co-exposure of C3G. The results indicate that C3G exerts protective effects on BPA-induced neurodevelopmental toxicity through improving transcription of neurogenesis related genes, enhancing antioxidative defense system and reducing cell apoptosis by regulation of apoptotic genes in zebrafish larvae. The results suggest that anthocyanins may play important role against the exogenous toxicity for vertebrates.

Evaluation of Anti-Venom Potential of Areca catechu Seed Extract on Bungarus caeruleus Venom

Areca catechu seeds and their extract/s are currently used to treat various ailments and infections including snakebites. The purpose of this investigation was to assess the inhibiting/neutralizing effect of ethyl acetate and aqueous ethanolic seed extracts of A. catechu on Bungarus caeruleus (krait) venom. The enzyme activities and their inhibition were evaluated using standard procedures (in vitro). In vivo studies were conducted using chick embryos and murine models. The extracts inhibited hyaluronidase and phospholipase A2 activities. Protease activity was neutralized by the aqueous ethanolic extract only. The IC50 value of aqueous ethanolic extract for hyaluronidase was 0.001 g/mL, while that for the ethyl acetate extract for phospholipase A2 was 0.006 g/mL. In addition, both the extracts neutralized the indirect hemolysis and fibrinogenolytic activity induced by B. caeruleus venom. The LD50 for the chick embryos was 4.9 µg/egg. The 50 and 100 µg aqueous ethanolic extracts neutralized the LD50 and the challenging dose (3LD50) of venom effectively in the chick embryo model. The LD50 of B. caeruleus venom in mice was 0.1927 µg/kg; the extract extended the survival time of the mice from 25 min to 30 and 35 min in 1:10 and 1:20 ((w/w) venom:extract) ratios, respectively. The extract also neutralized myotoxic activity. The A. catechu seed extract showed promising inhibitory properties against B. caeruleus venom. In this regard, academia and industries should work collaboratively to develop and formulate a cost-effective first-aid drug.