Whole-embryo Culture Research Articles

Recapitulating porcine cardiac development in vitro: from expanded potential stem cell to embryo culture models.

Domestic pigs (Sus scrofa) share many genetic, anatomical, and physiological traits with humans and therefore constitute an excellent preclinical animal model. Fundamental understanding of the cellular and molecular processes governing early porcine cardiogenesis is critical for developing advanced porcine models used for the study of heart diseases and new regenerative therapies. Here, we provide a detailed characterization of porcine cardiogenesis based on fetal porcine hearts at various developmental stages and cardiac cells derived from porcine expanded pluripotent stem cells (pEPSCs), i.e., stem cells having the potential to give rise to both embryonic and extraembryonic tissue. We notably demonstrate for the first time that pEPSCs can differentiate into cardiovascular progenitor cells (CPCs), functional cardiomyocytes (CMs), epicardial cells and epicardial-derived cells (EPDCs) in vitro. Furthermore, we present an enhanced system for whole-embryo culture which allows continuous ex utero development of porcine post-implantation embryos from the cardiac crescent stage (ED14) up to the cardiac looping (ED17) stage. These new techniques provide a versatile platform for studying porcine cardiac development and disease modeling.

Moderate alcohol consumption during pregnancy increases potency of two different drugs (the antifungal fluconazole and the antiepileptic valproate) in inducing craniofacial defects: prediction by the in vitro rat whole embryo culture

The prenatal exposure to ethanol (Eth), fluconazole (FLUCO) and sodium valproate (VPA) is related to effects on development, producing characteristic syndromic pictures. Among embryotoxic effects described for the three molecules, the alteration on craniofacial morphogenesis is a common feature in humans and animal models, including rodent embryos developed in vitro. The aim of the present work is to evaluate the developmental effects of low Eth serum concentration (17 mM, corresponding to the legal limit to drive in UK, USA, Canada, and many other countries) in mixture with increasing realistic concentrations of the antifungal drug FLUCO (62.5–500 µM) or with increasing realistic concentrations of the antiepileptic drug VPA (31.25–250 µM). Groups exposed to Eth alone (17–127.5 mM), FLUCO alone (62.5–500 µM) or VPA alone (31.25–750 µM) were also included. The chosen alternative animal model was the post-implantation rat whole embryo culture (WEC). E9.5 embryos were exposed in vitro to the test molecules during the whole test period (48 h, corresponding to the developmental stages characteristics of any vertebrate, for human embryos post-fertilization days 23–31). Data were statistically analyzed and processed for modelling applying the benchmark dose (BMD) and relative potency factor (RPF) approaches. Concentration-related effects on facial outcomes were observed in all experimental groups, with a significant enhancement in the groups co-exposed with Eth in comparison to the single exposures. Data obtained by the present work suggest an additional alert for the assumption of even low levels of alcohol in pregnant women during FLUCO or VPA therapy.

Teratogenic Effects of Organohalogen Contaminants Extracted from Whale Bacon in a Whole-Rat-Embryo Culture System.

Marine foods can be contaminated with organochlorines and the risk to human beings who consume these foods needs to be evaluated. We examined the teratogenic effects of contaminants extracted from whale bacon on rat embryos using a whole-embryo culture system. Embryonic day 11.5 embryos were cultured for 48 h with organohalogens extracted from whale bacon at low (polychlorinated biphenyls (PCBs): 0.32 ppm, dichlorodiphenyltrichloroethanes (DDTs): 0.16 ppm, chlordanes (CHLs): 0.02 ppm) and high (PCBs: 2.15 ppm, DDTs: 1.99 ppm, CHLs: 0.20 ppm) doses. The levels of organohalogen compounds in cultured embryos were determined. The organochlorine contaminants extracted from whale products were readily transferred to the cultured rat embryos. The number of heartbeats, yolk sac circulation score, and embryonic body circulation score of embryos did not change during the culture period in either exposure group. Cultured embryos treated with the low-dose contaminated medium for 48 h showed abnormalities of the mandible, and craniofacial or forelimb hematomas with an incidence of 50%. All embryos treated with the high-dose medium showed craniofacial abnormalities and cleft lip, and limb abnormalities and hematomas. These results indicate that the organohalogen contaminants in whale bacon may be teratogenic in a dose-dependent manner. Further studies are necessary to determine the dose–effect relationship.

Protocols for Investigating the Epithelial Properties of Cardiac Progenitor Cells in the Mouse Embryo.

Epithelial cardiac progenitor cells of the second heart field (SHF) contribute to growth of the vertebrate heart tube by progressive addition of cells from the dorsal pericardial wall to the cardiac poles. Perturbation of SHF development, including defects in apicobasal or planar polarity, results in shortening of the heart tube and a spectrum of congenital heart defects. Here, we provide detailed protocols for fixed section and wholemount immunofluorescence and live imaging approaches to studying the epithelial properties of cardiac progenitors in the dorsal pericardial wall during mouse heart development. Whole-embryo culture and electroporation methods are also presented, allowing for pharmacological and genetic perturbation of SHF development, as well as image analysis approaches to quantify cell features across the progenitor cell epithelium. These protocols are broadly applicable to the study of epithelia in the early embryo.

Grafting of Epiblast Stem Cell into the Epiblast and Whole-Embryo Imaging to Unveil Lineage Competence.

Here we describe a method to engraft epiblast stem cells (EpiSC) into the epiblast of gastrulation-stage mouse embryo to test the lineage propensity acquired by the EpiSCs during in vitro culture under different signaling conditions. After dissection and grafting, the recipient embryos can be grown in whole-embryo culture for up to 48h and the contribution of the EpiSC-derived cells to tissues in the recipient embryo is assessed by light sheet 3D microscopy.

Integrating in vitro chemical transplacental passage into a generic PBK model: A QIVIVE approach

With the increasing application of cell culture models as primary tools for predicting chemical safety, the quantitative extrapolation of the effective dose from in vitro to in vivo (QIVIVE) is of increasing importance. For developmental toxicity this requires scaling the in vitro observed dose-response characteristics to in vivo fetal exposure, while integrating maternal in vivo kinetics during pregnancy, in particular transplacental transfer. Here the transfer of substances across the placental barrier, has been studied using the in vitro BeWo cell assay and six embryotoxic compounds of different kinetic complexity. The BeWo assay results were incorporated in an existing generic Physiologically Based Kinetic (PBK) model which for this purpose was extended with rat pregnancy. Finally, as a “proof of principle”, the BeWo PBK model was used to perform a QIVIVE based on developmental toxicity as observed in various different in vitro toxicity assays. The BeWo results illustrated different transport profiles of the chemicals across the BeWo monolayer, allocating the substances into two distinct groups: the ‘quickly-transported’ and the ‘slowly-transported’. BeWo PBK exposure simulations during gestation were compared to experimentally measured maternal blood and fetal concentrations and a reverse dosimetry approach was applied to translate in vitro observed embryotoxicity into equivalent in vivo dose-response curves. This approach allowed for a direct comparison of the in vitro dose-response characteristics as observed in the Whole Embryo Culture (WEC), and the Embryonic Stem Cell test (cardiac:ESTc and neural:ESTn) with in vivo rat developmental toxicity data. Overall, the in vitro to in vivo comparisons suggest a promising future for the application of such QIVIVE methodologies for screening and prioritization purposes of developmental toxicants. Nevertheless, the clear need for further improvements is acknowledged for a wider application of the approach in chemical safety assessment.

Establishment of an in vitro method of rabbit embryo toxicity with toxicokinetics study.

This report introduces a novel method, rabbit whole embryo culture (WEC) combined with toxicokinetics (TK), for toxicity testing. Rodent WEC has been extensively used for in vitro screening of developmental toxicity. To improve the reliability of in vitro data, it is important to consider TK and species specificity. To test the utility and effectiveness of this method, we investigated the toxic effect of thalidomide on rabbit embryos and its behavior in test systems both in vitro and in vivo under the same experimental condition. The data showed that thalidomide induced embryo malformations such as embryonic brain hypoplasia, short limb buds, and declined embryonic growth both in vitro and in vivo. The toxic effect increased with the increasing exposure of the embryo to thalidomide. In addition, we observed similar toxic effects and exposure-effect relationships in vivo and in vitro. Therefore, we preliminarily conclude that this new method can effectively predict and explain thalidomide toxicity. Furthermore, we investigated the behavior of test compounds in the WEC system for the first time, and this method is expected to be an important technique for in vitro toxicity study after extensive verification.

MiR-322 treatment rescues cell apoptosis and neural tube defect formation through silencing NADPH oxidase 4.

AimsFailure of neural tube closure resulting from excessive apoptosis leads to neural tube defects (NTDs). NADPH oxidase 4 (NOX4) is a critical mediator of cell growth and death, yet its role in NTDs has never been characterized. NOX4 is a potential target of miR‐322, and we have previously demonstrated that miR‐322 was involved in high glucose‐induced NTDs. In this study, we investigated the effect of NOX4 on the embryonic neuroepithelium in NTDs and reveal a new regulatory mechanism for miR‐322 that disrupts neurulation by ameliorating cell apoptosis.MethodsAll‐trans‐retinoic acid (ATRA)‐induced mouse model was utilized to study NTDs. RNA pull‐down and dual‐luciferase reporter assays were used to confirm the interaction between NOX4 and miR‐322. In mouse neural stem cells and whole‐embryo culture, Western blot and TUNEL were carried out to investigate the effects of miR‐322 and NOX4 on neuroepithelium apoptosis in NTD formation.ResultsNOX4, as a novel target of miR‐322, was upregulated in ATRA‐induced mouse model of NTDs. In mouse neural stem cells, the expression of NOX4 was inhibited by miR‐322; still further, NOX4‐triggered apoptosis was also suppressed by miR‐322. Moreover, in whole‐embryo culture, injection of the miR‐322 mimic into the amniotic cavity attenuated cell apoptosis in NTD formation by silencing NOX4.ConclusionmiR‐322/NOX4 plays a crucial role in apoptosis‐induced NTD formation, which may provide a new understanding of the mechanism of embryonic NTDs and a basis for potential therapeutic target against NTDs.

Evaluation of in vitro embryotoxicity tests for Chinese herbal medicines

Chinese herbal medicines (CHMs) have been widely used during pregnancy, but feto-embryo safety tests are lacking. Here we evaluated in vitro embryotoxicity tests (IVTs) as alternative methods in assessing developmental toxicity of CHMs. Ten CHMs were selected and classified as strongly, weakly and non-embryotoxic. Three well validated IVTs and prediction models (PMs), including embryonic stem cell test (EST), micromass (MM) and whole embryo culture (WEC), were compared. All strongly embryotoxic CHMs were predicted by MM and WEC PM2. While all weakly embryotoxic CHMs were predicted by MM and WEC PM1. All non-embryotoxic CHMs were classified by EST, MM, but over-classified as weakly embryotoxic by WEC PM1. Overall predictivity, precision and accuracy of WEC determined by PM2 were better than EST and MM tests. Compared with validated chemicals, performance of IVTs for CHMs was comparable. So IVTs are adequate to identify and exclude embryotoxic potential of CHMs in this training set.

Decreased global DNA hydroxymethylation in neural tube defects: Association with polycyclic aromatic hydrocarbons

ABSTRACT5-Hydroxymethylcytosine (5hmC), a distinct epigenetic marker that plays a role in DNA active demethylation, has been reported to be important for embryonic development and may respond to environmental exposure. No studies have evaluated the association between DNA hydroxymethylation and the risk for fetal neural tube defects (NTDs), with consideration of prenatal exposure to polycyclic aromatic hydrocarbons (PAHs), a risk factor for NTDs.We measured the global levels of 5hmC% in neural tissue from 92 terminated NTD cases and 33 terminated non-malformed fetuses. A lower level of 5hmC% was found in the NTD cases (median [interquartile range]: 0.25 [0.12–0.39]) compared to the controls (0.45 [0.19–1.00]). After adjusting for periconceptional folate supplementation, risk for NTDs increased with decreasing tertiles of 5hmC% (odds ratio: 7.89, 95% confidence interval: 2.32, 26.86, for the lowest tertile relative to the top tertile; pfor trend = 0.002). Linear regression revealed that concentrations of high-molecular-weight PAHs (H_PAHs) in fetal liver tissue were negatively associated with log2-transformed 5hmC%. Superoxide dismutase activity and 5hmC% were positively correlated in fetal neural tissue (rs = 0.64; p < 0.05). A mouse whole-embryo culture model was used for further validation. Decreased levels of 5hmC% and increased levels of reactive oxygen species were found in mouse embryos treated with BaP, a well-studied PAH. Taken together, levels of 5hmC% in fetal neural tissue were inversely associated with the risk for NTDs, and this association may be related to oxidative stress induced by exposure to PAHs.

Teratogenic Potential of Traditionally Formulated and Nano-Encapsulated Vitamin A in Two Vertebrate Models, Rattus norvegicus and Xenopus Laevis

Nano-encapsulation is applied for the preparation of functional food to preserve micronutrients degradation and to ameliorate their absorption. Being nano-encapsulation already related to increased vitamin A embryotoxicity, we aimed to evaluate the effect of traditionally formulated (BULK-A) and nano-encapsulated vitamin A (NANO-A) in two different vertebrate models: rat post implantation Whole Embryo Culture (WEC) and Frog Embryo Teratogenesis Assay-Xenopus (FETAX). After benchmark-dose modelling, WEC results showed that NANO-A was 7 times more effective than BULK-A, while FETAX results indicated that X. laevis development was affected only by NANO-A. The relative potency of WEC was 14 times the potency of FETAX, suggesting a minor role of preformed vitamin A in X. laevis development in respect to mammal embryogenesis. Results from this work prompt the necessity to monitor the use of food supplemented with NANO A, since even low doses can elicit teratogenic effects on vertebrate embryos due to its increased bioavailability.

Rabbit Whole Embryo Culture.

The rabbit is a mainstay of regulatory developmental toxicity testing; however, due to the historic absence of experimental tools for this species, there is a dearth of information about its fundamental embryology and the mechanisms underlying developmental toxicity. Relatively recently, there have been advances in the methods of rabbit whole embryo culture (WEC), and this has prompted an increase in understanding of rabbit embryogenesis. Described herein are the methods used to remove early somite-stage embryos (gestation day 9) and sustain their growth for 48h. Although there are similarities to the well-described rodent WEC, there are also important differences. Akin to rodent WEC, the major phases of organogenesis can be investigated, including neural tube development, cardiac looping, segmentation, and the development of the anlagen of the optic and otic regions, craniofacial development, somites, and early limb bud development. Unlike the rodent, rabbit WEC requires the use of an apparatus that allows for the continuous gassing of embryos, and one may observe the expansion and closure of the visceral yolk sac around the embryo. After completion of the culture period, embryos are examined across several growth and developmental parameters including a quantitative morphological scoring system. Embryonic growth and development in the absence of maternal influences allows for the study of the direct action of agents or their metabolites on the embryo. The use of both rodent and rabbit WEC together is a powerful strategy with which to investigate species-specific vulnerabilities to specific agents.

Rat Whole Embryo Culture.

The direct effects of chemical exposures, environmental extremes, and nutrient quality/quantity have been very difficult to study in mammalian embryos due to their anatomical inaccessibility, paucity of tissue, and other factors that make human studies unethical. Many acute and chronic developmental anomalies can trace their origins to postimplantation phases of gestation, where the organs are first being established and growth and differentiation are in highly active states of flux. Most chemical insults and conditions that produce birth defects are believed to act during this period of organogenesis. The evolution of rodent whole embryo culture (WEC) techniques has provided a valuable experimental model where physiological conditions and exposures can be carefully controlled and manipulated to test hypotheses and explore biochemical and molecular mechanisms of action that would otherwise be extremely difficult. Exposure to chemicals can be controlled through their direct addition to the culture medium. Optimal in vitro culture conditions support the growth of intact, viable conceptuses (embryo and associated extraembryonic membranes) from early egg cylinder stages through the establishment of the neural plate, gastrulation, neural tube closure, onset of active heartbeat and circulation, and the initial formation of all major organ systems that occur prior to the establishment of a functional placenta. Detailed comparisons of in vivo and in vitro growth show that conceptuses grown in WEC are nearly identical, structurally and functionally, to conceptuses of the same developmental stage that are allowed to develop normally in utero during a comparable developmental period. Culture conditions and mechanical apparatuses can be modified to suit a large number of different experimental approaches and paradigms.

Hedgehog signaling dynamics in mouse embryos determined by a bioluminiscent reporter.

Determination of cellular signaling in live embryos is key to understand the molecular processes that drive development. Here, we show that a transgenic mouse line carrying a luciferase-based gene reporter of Gli-mediated transcriptional activation (Gli-Luc) displays sonic hedgehog (Shh) signaling in discrete developmental processes during short-term cultures of whole embryos or embryo explants. The bioluminescence in E9.5 embryos was detected in regions in which Shh activity has been demonstrated. Later, in E10.5 embryos, bioluminescence intensity markedly increased, mostly corresponding to the high Shh activity of the developing midbrain and limb. Notably, the dynamic range of the Gli-Luc reporter in the developing limb revealed the progressive emergence of bioluminescence in the zone of polarizing activity, where reporter activity locally increased and spatially spread in agreement with the signaling gradient expected for Shh. In the midbrain of E9.5 mouse embryos, bioluminescence was not detected along the ventral region as expected but, instead, Shh-dependent anterior and posterior bioluminescence foci emerged by E10.5 indicating that the Gli-Luc reporter can only respond transcriptionally to relatively high levels of GliA and/or without the interaction with other transcription factors. The present work supports the use of bioluminescence to identify and study the dynamics of centers of morphogen signaling during mouse embryogenesis.

Relative potency ranking of azoles altering craniofacial morphogenesis in rats: An in vitro data modelling approach

Facial malformations represent one of the most frequent abnormality in humans. The adverse outcome pathway involved in facial defects seems to be related to retinoic acid (RA) pathway imbalance. Environmental agents inducing craniofacial malformations in experimental models include pesticides (especially azole fungicides). By using the in vitro alternative method postimplantation rat whole embryo culture (WEC), we evaluated the intrinsic embryotoxic activity of some azole antifungals (cyproconazole, CYPRO; triadimefon, FON; flusilazole, FLUSI; and prochloraz, PCZ), in comparison to RA. All the tested molecules induced in a dose-related manner specific defects of the craniofacial structures (fused branchial arches), similar to those induced by RA. Collected data were modelled using PROAST 65.5 software to characterise the relative potency factors (RPFs) versus RA. In comparison to RA, all the evaluated azoles were less potent, showing among them a similar potency. Our data suggest a possible azole-related RA signalling perturbation to be further investigated. Moreover, the present results indicate the approach used in this work to be an interesting tool applicable to the hazard evaluation of novel compounds or the assessment of combined exposure to azoles or other dismorphogens.

A comparison of the embryonic stem cell test and whole embryo culture assay combined with the BeWo placental passage model for predicting the embryotoxicity of azoles

In the present study, we show the value of combining toxico-dynamic and -kinetic in vitro approaches for embryotoxicity testing of azoles. Both the whole embryo culture (WEC) and the embryonic stem cells test (EST) predicted the in vivo potency ranking of twelve tested azoles with moderate accuracy. Combining these results with relative placental transfer rates (Papp values) as determined in the BeWo cell culture model, increased the predictability of both WEC and EST, with R2 values increasing from 0.51 to 0.87 and from 0.35 to 0.60, respectively. The comparison of these in vitro systems correlated well (R2 = 0.67), correctly identifying the in vivo strong and weak embryotoxicants. Evaluating also specific gene responses related with the retinoic acid and sterol biosynthesis pathways, which represent the toxicological and fungicidal mode of action of azoles respectively in the WEC and EST, we observed that the differential regulation of Dhrs3 and Msmo1 reached higher magnitudes in both systems compared to Cyp26a1 and Cyp51. Establishing sensitive biomarkers across the in vitro systems for studying the underlying mechanism of action of chemicals, such as azoles, is valuable for comparing alternative in vitro models and for improving insight in the mechanism of developmental toxicity of chemicals.

Manipulation of MicroRNAs in Cultured Mouse Embryos: Applications for Developmental Toxicology.

MicroRNAs (miRNAs) are a class of small noncoding RNAs that modulate the expression of virtually all genes. miRNAs have been implicated in the regulation of a broad range of cellular and physiologic processes. Dysregulation of miRNAs has been found to be associated with a variety of diseases and disorders, including teratogenesis. One of the important strategies to reveal miRNA function in teratogenesis is the manipulation of their expression levels and patterns in specific regions in the developing embryos. The combination of microinjection technology with the whole embryo culture (WEC) system provides an excellent platform for the mechanistic study of the roles of miRNAs in teratogenesis. Here we describe an approach to investigating and manipulating miRNAs in cultured mouse embryos and present protocols for whole embryo culture, microinjection of miRNA mimics or inhibitors, validation of the efficacy of microRNA modulation in embryos, and assessment of the teratogen-induced apoptosis.

Contributions of the chick embryo and experimental embryology to understanding the cellular mechanisms of neurulation.

The chick embryo has served as a workhorse for experimental embryological studies designed to elucidate mechanisms underlying neurulation, the process that forms the neural tube, the rudiment of the entire adult central nervous system. Early chick embryos developing in whole-embryo culture can be readily manipulated in cut-and-paste-type experiments, and this attribute makes this model system unparalleled for studying the morphogenesis of embryos and their organ rudiments. How the chick embryo and experimental embryology have contributed to our understanding of critical events of neurulation are summarized.

A transcriptomic approach for evaluating the relative potency and mechanism of action of azoles in the rat Whole Embryo Culture

We evaluated the effect of six azoles on embryonic development in the rat whole embryo culture (WEC). Using the total morphological scoring system (TMS), we calculated the ID10 concentration (effective dose for 10% decrease in TMS). For evaluating gene specific responses, we combined previously and newly collected transcriptomics data of rat WEC exposed to a total of twelve azoles at their ID10 for 4h. Results revealed shared expressions responses in genes involved in the retinoic acid (RA) and sterol biosynthesis pathways, which are respectively representatives of developmental toxicity and targeted fungicidal action of the azoles. Azoles with more pronounced effects on the regulation of RA-associated genes were generally characterized as more potent embryotoxicants. Overall, compounds with strong sterol biosynthesis related responses and low RA related responses were considered as more favourable candidates, as they specifically regulated genes related to a desired target response. Among the identified sterol associated genes, we detected that methylsterol monooxygenase 1 (Msmo1) was more sensitively induced compared to Cyp51, a classical biomarker of this pathway. Therefore, we suggest that Msmo1 could be a better biomarker for screening the fungicidal value of azoles. In summary, we conclude that the embryonic regulation of RA and sterol metabolic pathways could be indicators for ranking azoles as embryotoxicants and determining their drug efficacy.

In vitro to in vivo extrapolation of effective dosimetry in developmental toxicity testing: Application of a generic PBK modelling approach

Incorporation of kinetics to quantitative in vitro to in vivo extrapolations (QIVIVE) is a key step for the realization of a non-animal testing paradigm, in the sphere of regulatory toxicology. The use of Physiologically-Based Kinetic (PBK) modelling for determining systemic doses of chemicals at the target site is accepted to be an indispensable element for such purposes. Nonetheless, PBK models are usually designed for a single or a group of compounds and are considered demanding, with respect to experimental data needed for model parameterization. Alternatively, we evaluate here the use of a more generic approach, i.e. the so-called IndusChemFate model, which is based on incorporated QSAR model parametrization. The model was used to simulate the in vivo kinetics of three diverse classes of developmental toxicants: triazoles, glycol ethers' alkoxyacetic acid metabolites and phthalate primary metabolites. The model required specific input per each class of compounds. These compounds were previously tested in three alternative assays: the whole-embryo culture (WEC), the zebrafish embryo test (ZET), and the mouse embryonic stem cell test (EST). Thereafter, the PBK-simulated blood levels at toxic in vivo doses were compared to the respective in vitro effective concentrations. Comparisons pertaining to relative potency and potency ranking with integration of kinetics were similar to previously obtained comparisons. Additionally, all three in vitro systems produced quite comparable results, and hence, a combination of alternative tests is still preferable for predicting the endpoint of developmental toxicity in vivo. This approach is put forward as biologically more plausible since plasma concentrations, rather than external administered doses, constitute the most direct in vivo dose metric.